© 2016 Robert Wilson Powell III

IMPROVING THE THERMOSTABILITY AND INCREASING THE SUBSTRATE RANGE OF OLD YELLOW ENZYME HOMOLOGS AND AMINOLEVULINIC ACID SYNTHASE

THROUGH PROTEIN ENGINEERING

By

ROBERT WILSON POWELL III

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2016

“To my family and friends”

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ACKNOWLEDGMENTS

I would like to acknowledge my faculty advisor Dr. Jon Stewart for his guidance. I

am very grateful to him for the opportunity to do work in his lab. I feel fortunate to have

been able to work on projects which I enjoyed so much. I am very proud of the work we

do in this lab and am grateful to have been able to contribute what I could during my

time. I would like to acknowledge the University of Florida. And, I would like to

acknowledge the NSF for funding my research.

I would like to acknowledge Dr. Bradford Sullivan for his assistance during the

OYE 1 Y196 project. I would like to acknowledge Athena Patterson for her assistance

during the OYE2.6 Y78Xsm / I113Xsm project. I would like to acknowledge Steven

Crichton for all his work during the OYE2.6 Y78Xsm / I113Xsm and OYE2.6 Y78Xsm /

I113C / F247X projects. I would like to acknowledge Dr. Filip Boratynski for all of his

work during the OYE 2.6 thermostability project. I would like to acknowledge Matthew

Burg and Dr. Steven Bruner for all of their help during the OYE 3 crystallization project. I

would like to acknowledge Steven Crichton again for all his work during the mALAS

project. Every one of these people have contributed to these projects in a meaningful

way and I am grateful to them all for it.

I would like to thank Dr. Hillary Lathrop for all her help with the Beckman and

Gilson HPLCs as well as for all her work maintaining our instrument room. I would like

to acknowledge Sarah Franz for all her help with the MSTFA derivatization protocols. I

would like to thank Louis Mouterde for all his work with the acyl-CoA separation method

development. I am grateful for their help running and maintaining the lab. I would like to

thank my family and friends for their support. I would especially like to thank my mom,

Janet Powell, for all of her support during my time here. And, I would also like to thank

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Nicole Gibbons for her love and support. I am very thankful for all the help Nicole has

given me during our time here. I am very grateful to have met her and to have been able

to go on this journey with her.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF TABLES .......................................................................................................... 11

LIST OF FIGURES ........................................................................................................ 12

LIST OF ABBREVIATIONS ........................................................................................... 19

ABSTRACT ................................................................................................................... 20

CHAPTER

1 PROBING POSITION Y196 IN OLD YELLOW ENZYME 1 .................................... 22

Background ............................................................................................................. 22

Isolation and Purification of OYE ...................................................................... 22 Catalytic cycle and substrates of OYE ............................................................. 23 Structure and Mechanism of OYE .................................................................... 24

Project Overview .............................................................................................. 25 Results and Discussion........................................................................................... 25

OYE 1 YI96 Site-Saturation Mutagenesis Library ............................................. 25 Crystal Structure of OYE 1 YI96C .................................................................... 27

Experimental ........................................................................................................... 28 General............................................................................................................. 28 Cloning ............................................................................................................. 29

Construction of plasmids used to make the OYE 1 Y196 library ................ 29 Construction of mutants in the OYE 1 Y196X randomized library .............. 29

Substrates ........................................................................................................ 30 2-(Hydroxymethyl)-cyclopent-2-enone (1) .................................................. 30 2-(Hydroxymethyl)-cyclohex-2-enone (2) ................................................... 31

Methyl 2-(hydroxymethyl)acrylate (3) ......................................................... 31 (S)-(+)-Carvone (7) .................................................................................... 31

(R)-(-)-Carvone (8) ..................................................................................... 32

2-Methyl-2-cyclopenten-1-one (11) ............................................................ 32

2-Methyl-2-cyclohexen-1-one (12) ............................................................. 32 Screening Assay .............................................................................................. 33 Protein Purification and Crystallogenesis of Y196C OYE 1 .............................. 33 The Data Collection and Crystal Structure of OYE 1 Y196 Mutants ................. 35

Conclusions ............................................................................................................ 36

2 IMPROVING THE PRODUCT RANGE OF OYE 1 AND OYE 2.6 THROUGH PROTEIN ENGINEERING ...................................................................................... 61

Background ............................................................................................................. 61

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Mutagenesis of the East Side of the Active Site in OYE ................................... 61

Project Summary .............................................................................................. 64 Results and Discussion........................................................................................... 65

OYE 2.6 Y78Xsm / I113Xsm Randomized Library .............................................. 65 OYE 2.6 Y78Xsm / I113Xsm Presequenced Library ........................................... 68 OYE 2.6 Y78Xsm / I113C / F247X Randomized Libraries ................................. 68

Experimental ........................................................................................................... 70 General............................................................................................................. 70

Cloning ............................................................................................................. 70 Construction of plasmids used to make the OYE 2.6 ................................. 70 Construction of the mutants in the OYE 2.6 Y78Xsm / I113Xsm

randomized library .................................................................................. 70 Construction of the mutants in the OYE 2.6 Y78Xsm-I113Xsm

presequenced library .............................................................................. 73 Addressing concatomeric primer inserts .................................................... 74

Construction of mutants in the OYE 2.6 Y78Xsm / I113C / F247X randomized libraries ............................................................................... 74

Substrates ........................................................................................................ 75 Methyl 2-(hydroxymethyl)acrylate (1) ......................................................... 75

2-(Hydroxymethyl)-cyclohex-2-enone (2) ................................................... 76 2-(Hydroxymethyl)-cyclopent-2-enone (3) .................................................. 76

Screening Assay .............................................................................................. 76 Conclusions ............................................................................................................ 77

3 IMPROVING THE THERMOSTABILITY OF OYE 2.6 THROUGH PROTEIN ENGINEERING ....................................................................................................... 95

Background ............................................................................................................. 95

Improving Thermostability through Mutagenesis .............................................. 95 Project Summary .............................................................................................. 97

Results and Discussion........................................................................................... 98 Residues with High Local B-Factors ................................................................. 98 Dimer Interface Residues ............................................................................... 100

Combining Thermostabilizing Mutations ......................................................... 100 Crystallization of OYE 2.6 D141E-S388P ....................................................... 101

Experimental ......................................................................................................... 104 General........................................................................................................... 104

Cloning ........................................................................................................... 104 Construction of the plasmid used as a template for the OYE 2.6 thermal

stability libraries .................................................................................... 104 Construction of OYE 2.6 libraries ............................................................. 105

Substrates ...................................................................................................... 106

2-Methyl-2-cyclopenten-1-one (1) ............................................................ 106 Screening ................................................................................................. 107

The Protein Purification and Crystallization of OYE 2.6 Mutants .................... 109 Data Collection and Structure Solution of OYE 2.6 D141E-S388P ................ 112 B-Factor Data and Statistics ........................................................................... 113

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Conclusions .......................................................................................................... 113

4 THE STRUCTURE OF Saccharomyces cerevisiae OLD YELLOW ENZYME 3 ... 135

Background ........................................................................................................... 135

Crystallization of OYE Family Members ......................................................... 135 Project Summary ............................................................................................ 136

Results and Discussion......................................................................................... 137 Crystallization of OYE 3.................................................................................. 137 Data Reduction and Structure Solution .......................................................... 139

OYE 3 W116 Site Saturation Mutagenesis ..................................................... 143 X-Ray Crystallography Studies of OYE 3 W116 Mutants and Related

Proteins ....................................................................................................... 144

Experimental ......................................................................................................... 146 General........................................................................................................... 146 Cloning ........................................................................................................... 146

Construction of plasmid used for libraries ................................................ 146 Construction of an OYE 3 W116 site-saturation mutagenesis library ....... 147

Testing of Phenol Binding to OYE 3 ............................................................... 149 GST-OYE 3 Fusion Protein Purification and Crystallogenesis ....................... 149 Native OYE 3 Protein Purification and Crystallogenesis ................................ 150

Alkene Substrate for OYE 3 ........................................................................... 154 2-(Hydroxymethyl)-cyclopent-2-enone (1) ................................................ 154

2-(Hydroxymethyl)-cyclohex-2-enone (2) ................................................. 154 (R)-Pulegone (3) ...................................................................................... 155 (S)-(+)-Carvone (4) .................................................................................. 155 (R)-(-)-Carvone (5) ................................................................................... 156

2-Methyl-2-cyclopenten-1-one (11) .......................................................... 156

2-Methyl-2-cyclohexen-1-one (12) ........................................................... 156 3-Methyl-cyclohexen-1-one (13) .............................................................. 157

3-Ethyl-cyclohexen-1-one (14) ................................................................. 157 3-Methyl-cyclopenten-1-one (15) ............................................................. 157 4-Ethyl-4-methyl-2-cyclohexen-1-one (21) ............................................... 158

4-Isoproply-4-methyl-2-cyclohexen-1-one (22) ........................................ 158 4,4-Diethyl-2-cyclohexen-1-one (23) ........................................................ 158 Spiro[5.5]undec-1-en-3-one (24) .............................................................. 159 2-Butylidenecyclohexanone (25) .............................................................. 159

Screening ....................................................................................................... 159 Conclusions .......................................................................................................... 160

5 IMPROVING THE SUBSTRATE RANGE OF AMINOLEVULINIC ACID SYNTHASE THROUGH PROTEIN ENGINEERING............................................. 190

Background ........................................................................................................... 190

Positions of Interest ........................................................................................ 191 Threonine 148 .......................................................................................... 191

Isoleucine 151 .......................................................................................... 191

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Arginine 85 ............................................................................................... 192

The Glycine Loop ........................................................................................... 192 Project Overview ............................................................................................ 193

Results and Discussion......................................................................................... 195 Detecting δ-AL-pyrrole Compounds with Ehrlich’s Reagent ........................... 195 Preparation and Detection of Succinyl-CoA ................................................... 196 In Situ Succinyl-CoA Formation ...................................................................... 196

Detecting Amino Products Using PITC and MSTFA Derivatives .................... 197

mALAS R85, T148 and I151 Site-Saturation Mutagenesis Libraries .............. 199 Experimental ......................................................................................................... 199

General........................................................................................................... 199 Cloning ........................................................................................................... 200

Construction of plasmid used to make ALAS libraries ............................. 200

Construction of ALAS libraries ................................................................. 200 Preparation and Detection of Succinyl-CoA ................................................... 202

Succinyl-CoA Regeneration System .............................................................. 203

Detecting δ-AL-Pyrrole Compounds with Ehrlich’s Reagent .......................... 204 Derivatizing Amino Acids Using PITC ............................................................ 205 Detecting PITC Amino Acid Derivatives by HPLC .......................................... 206

Derivatizing Amino Acids Using MSTFA......................................................... 206 Detecting MSTFA Amino Acid Derivatives Using GC-MS .............................. 207

Conclusions .......................................................................................................... 207

APPENDIX

A LIST OF PRIMERS ............................................................................................... 223

Chapter 1 Primers ................................................................................................. 223 Chapter 2 Primers ................................................................................................. 224

Chapter 3 Primers ................................................................................................. 225 Chapter 4 Primers ................................................................................................. 226

Chapter 5 Primers ................................................................................................. 228

B MUTAGENIC PLASMIDS ..................................................................................... 232

C PLASMID SEQUENCES ....................................................................................... 233

Sequence of pET3b-OYE1 ................................................................................... 233

Sequence of pBS2 ................................................................................................ 235 Sequence of pFB1 ................................................................................................ 238 Sequence of pRP4 ................................................................................................ 241

Sequence of pGF23 .............................................................................................. 243

D GC AND HPLC METHODS .................................................................................. 246

AZW2.Meth ........................................................................................................... 246 AZW3.Meth ........................................................................................................... 246 BTS2.Meth ............................................................................................................ 247

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BTS3.Meth ............................................................................................................ 247

BTS4.Meth ............................................................................................................ 248 BTS7.Meth ............................................................................................................ 248

BTS8.Meth ............................................................................................................ 249 FB1.Meth .............................................................................................................. 249 JON.Meth .............................................................................................................. 250 SEF.Meth .............................................................................................................. 250 YAP.Meth .............................................................................................................. 251

LMM.Meth ............................................................................................................. 251 RWP2.Meth .......................................................................................................... 252

E PLASMID MAPS ................................................................................................... 253

pET3b-OYE .......................................................................................................... 253 pBS2 ..................................................................................................................... 253 pFB1 ..................................................................................................................... 254

pRP4 ..................................................................................................................... 254 pGF23 ................................................................................................................... 255

LIST OF REFERENCES ............................................................................................. 256

BIOGRAPHICAL SKETCH .......................................................................................... 263

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LIST OF TABLES

Table page 2-1 List of the presequenced alkene reductase libraries ........................................... 79

2-2 OYE 2.6 best variants discovered during the OYE 2.6 ISM studies ................... 80

2-3 Q scores for NNK randomized libraries. ............................................................. 80

3-1 Crystallographic data collection and refinement statistics ................................ 116

3-2 Q scores for NNK randomized libraries. ........................................................... 117

4-1 Crystallographic data collection and refinement statistics. ............................... 162

4-2 Crystallographic data collection and refinement statistics. ............................... 163

4-3 Distances between the β-carbon of each active site residue to the ligand ....... 164

5-1 Retention times of PIT-amino acid derivative standards from HPLC ................ 209

A-1 List of mutagenic primers for chapter 1. ........................................................... 223

A-2 List of mutagenic and sequencing primers for chapter 2 .................................. 224




B-1 Mutagenic plasmids used in this study. ............................................................ 232

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LIST OF FIGURES

Figure page 1-1 The catalytic cycle of G6PDH investigated by Warburg and Christian ............... 38

1-2 The catalytic cycle of OYE 1 established by Massey and Vas ........................... 38

1-3 List of OYE 1 substrates from the literature ........................................................ 39

1-4 FMN Diagram displaying the FMN environment in the active site of OYE 1 ....... 50

1-5 Catalytic mechanism of OYE 1 ........................................................................... 50

1-6 OYE substrate binding modes ............................................................................ 51

1-7 OYE 1 active site diagram .................................................................................. 52

1-8 List of Baylis-Hillman substrates screened by OYE 1 Y196 library ..................... 53

1-9 List of carvone substrates screened by OYE 1 Y196 library ............................... 54

1-10 List of screening substrates screened by OYE 1 Y196 library ............................ 55

1-11 Calculations for obtaining a Qscore of a pooled plasmid mix from a NNK primer mix using data from a theoretical sequencing electropherogram ....................... 56

1-12 OYE 1 Y196 library screening results for 1 ......................................................... 57





1-17 OYE 1 Y196 library screening results for 11 ....................................................... 59

1-18 OYE 1 Y196 library screening results for 12 ....................................................... 60

1-19 Alignment of OYE 1 wt. and OYE 1 Y196C ........................................................ 60

2-1 Flipped binding mode ......................................................................................... 81

2-2 List of Chapter 2 substrates ................................................................................ 82

2-3 (S)-(+)-carvone bound in a flipped binding mode to the active site of OYE 1 W116I ................................................................................................................. 83

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2-4 Mechanism of OYE 2.6 ....................................................................................... 83

2-5 Diagram of the residues in the OYE 2.6 active site ............................................ 84

2-6 Malonate bound in the active site of OYE 2.6 Y78W-I113C ............................... 84

2-7 Substrate 1 and 2 modeled into the active of OYE 2.6 Y78W and OYE 2.6 wt .. 85

2-8 Sequence alignment of OYE 2.6 with a sample containing primer inserts .......... 85

2-9 Best variants discovered during both the ISM project and this project using a small residue matrix for obtaining the (R)-5 product from substrate 2 ................ 86

2-10 OYE 2.6 Y78Xsm / I113Xsm library screening results for substrate 2 ................ 87

2-11 OYE 2.6 Y78A / I113C / F247X screening results for substrate 2 ...................... 88

2-12 OYE 2.6 Y78C / I113C / F247X screening results for substrate 2 ...................... 89

2-13 OYE 2.6 Y78G / I113C / F247X screening results for substrate 2 ...................... 90

2-14 OYE 2.6 Y78S / I113C / F247X screening results for substrate 2 ...................... 91

2-15 OYE 2.6 Y78T / I113C / F247X screening results for substrate 2....................... 92

2-16 OYE 2.6 Y78V / I113C / F247X screening results for substrate 2 ...................... 93

2-17 Calculations for obtaining a Qscore of a pooled plasmid mix from a KST primer mix using data from a theoretical sequencing electropherogram ....................... 94

3-1 The fraction of B-factors for each position over the average B-factors of all positions in the structure ................................................................................... 118

3-2 The relative B-factors of all three published OYE 2.6 wild type structures ....... 118

3-3 The positions targeted during the ISM thermostability project, their region in the protein, and their B-factors for structure 3TJL ............................................ 119

3-4 The positions targeted during the local maximum project, their region in the protein, and their B-factors for structure 3TJL .................................................. 119

3-5 The B-factor values for the positions targeted during the local maximum project ............................................................................................................... 120

3-6 The positions selected for mutagenesis during the dimer interface project ...... 120

3-7 The results for the small scale screening of the OYE 2.6 S388 library testing all 19 possible replacements with substrate 1 .................................................. 121

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3-8 The results of screening the OYE 2.6 E41X NNK randomized library .............. 121

3-9 The results of screening the OYE 2.6 D141X NNK randomized library ............ 122

3-10 The results of screening the OYE 2.6 E145X NNK randomized library ............ 122

3-11 The results of screening the OYE 2.6 K330X NNK randomized library ............ 123

3-12 The results of screening the OYE 2.6 I214X NNK randomized library .............. 123

3-13 The results of screening the OYE 2.6 W244X NNK randomized library ........... 124

3-14 The results of screening the OYE 2.6 L260X NNK randomized library ............. 124

3-15 The results of screening the OYE 2.6 F307X NNK randomized library ............ 125

3-16 The results of screening the OYE 2.6 I311X NNK randomized library .............. 125

3-17 The results from the large scale screening assay............................................. 126

3-18 The results of the best mutants from all ten ISM-libraries ................................. 127

3-19 The results of the best mutants from OYE 2.6 E41X ........................................ 127

3-20 The results of the best mutants from OYE 2.6 D141X ...................................... 128

3-21 The results of the best mutants from OYE 2.6 E145X ...................................... 128

3-22 The results of the best mutants from all projects .............................................. 129

3-23 The positions targeted in both the ISM thermostability and local maximum projects ............................................................................................................. 130

3-24 The relative B-factor fractions from the OYE 2.6 D141E-S388P structure ....... 130

3-25 The relative B-factor fraction of all OYE 2.6 positions along the 3UPW structure ........................................................................................................... 131

3-26 The relative B-factor fraction of all OYE 2.6 positions along the 3TJL structure ........................................................................................................... 131

3-27 The B-factor fractions for all three published OYE 2.6 wt. structures ............... 132

3-28 The B-factor fractions of both the three OYE 2.6 wt. structures and the OYE 2.6 D141E-S388P structure .............................................................................. 132

3-29 The relative B-factor fractions of OYE 2.6 wt. in structure 3TJL ....................... 133

3-30 The relative B-factor fractions of OYE 2.6 D141E-S388P structure .................. 133

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3-31 The regeneration system used to make NADPH which reduces the FMN of OYE and allows the protein to turnover ............................................................ 134

4-1 Schematic illustration of the FMN environment in the active site of OYE homologs .......................................................................................................... 165

4-2 The mechanism of OYE 3 ................................................................................ 165

4-3 Diagram of the positions in the active site of OYE homologs ........................... 166

4-4 Loop 6 in OYE homologs .................................................................................. 167

4-5 List of OYE 3 substrates and reported conversion from the literature .............. 168

4-6 First set of substrates and theoretical binding mode products .......................... 177

4-7 Second set of substrates and theoretical binding mode products..................... 178

4-8 Third set of substrates and theoretical binding mode products......................... 179

4-9 The reactions used to test phenol binding by OYE 3 ........................................ 180

4-10 Crystals and crystallization conditions for of OYE 1, OYE 2.6, and OYE 3 ...... 180

4-11 The structure of OYE 3 ..................................................................................... 181

4-12 The active site for both OYE 1 and OYE 3 with bound FMN and substrate ..... 181

4-13 The active site for both OYE 3 and OYE 3 W116V with bound FMN and p-HBA .................................................................................................................. 182

4-14 The active site for both OYE 1 and OYE 1 F296S with bound FMN and p-HBA .................................................................................................................. 182

4-15 Results from screening the OYE 3 W116 site-saturation library against substrate 1 ........................................................................................................ 183





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4-20 Results from screening the OYE 3 W116 site-saturation library against substrate 11 ...................................................................................................... 185









5-1 The reaction of glycine and succinyl-CoA to make δ-AL using ALAS as a catalyst ............................................................................................................. 210

5-2 The proposed mechanism of ALAS .................................................................. 211

5-3 The active site of ALAS from R. capsulatus with glycine bound to PLP ........... 212

5-4 The active site of ALAS from R. capsulatus with succinyl-CoA ........................ 212

5-5 Reaction scheme of the derivatizing of δ-AL-pyrrole with Ehrlich’s reagent ..... 213

5-6 Reaction scheme of succinic anhydride with CoA to make succinyl-CoA ......... 213

5-7 The coupling of ALAS production of CoA from succinyl-CoA to α-Ketoglutarate Dehydrogenase production of NADH from NAD+ ....................... 214

5-8 Derivatization of amino acids using PITC ......................................................... 215

5-9 Derivatization of amino acids using MSTFA ..................................................... 215

5-10 The results for the reaction of δ-AL-pyrrole with Ehrlich’s reagent ................... 215

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5-11 The results for the reaction of δ-AL-pyrrole with Ehrlich’s reagent using a plate reader ...................................................................................................... 216

5-12 The reaction of succinyl-CoA with hydroxyl amine to displace the CoA. .......... 217

5-13 HPLC results from the reaction of succinic anhydride and CoA to make succinyl-CoA..................................................................................................... 217

5-14 HPLC results from the synthesis of succinyl-CoA from succinic anhydride and CoA co-eluted with 10x CoA ...................................................................... 218

5-15 HPLC results from the reaction of succinyl-CoA with hydroxylamine ............... 219

5-16 Results of the succinyl-CoA regeneration system ............................................ 220

5-17 Results of reactions from the succinyl-CoA regeneration system using mALAS and mALAS R433K ............................................................................. 221

5-18 Results of amino acid derivatization with MSTFA ............................................. 222

D-1 AZW2.Meth....................................................................................................... 246

D-2 AZW3.Meth....................................................................................................... 246

D-3 BTS2.Meth........................................................................................................ 247

D-4 BTS3.Meth........................................................................................................ 247

D-5 BTS4.Meth........................................................................................................ 248

D-6 BTS7.Meth........................................................................................................ 248

D-7 BTS8.Meth........................................................................................................ 249

D-8 FB1.Meth .......................................................................................................... 249

D-9 JON.Meth ......................................................................................................... 250

D-10 SEF.Meth ......................................................................................................... 250

D-11 YAP.Meth ......................................................................................................... 251

D-12 LMM.Meth......................................................................................................... 251

D-13 RWP2.Meth ...................................................................................................... 252

E-1 pET3b-OYE ...................................................................................................... 253

E-2 pBS2 ................................................................................................................. 253

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E-3 pFB1 ................................................................................................................. 254

E-4 pRP4 ................................................................................................................ 254

E-5 pGF23 .............................................................................................................. 255

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LIST OF ABBREVIATIONS

FPLC Fast Protein Liquid Chromatography

GC-FID Gas Chromatography Flame Ignition Detector

GC-MS Gas Chromatography Mass Spectrometry

HPLC High Pressure Liquid Chromatography

RMSD Root Mean Square Deviation

Y78X Y78 represents tyrosine at position 78 in the sequence of a protein. The X represents a random set of amino acids at that position that can include any, of the 20 amino acids.

Y78XKST Y78 represents tyrosine at position 78 in the sequence of a protein. The XKST represents a mutation at that position to a set of random amino acids which were obtained from a KST codon mix: alanine, cysteine, glycine, and serine.

Y78Xsm Y78 represents tyrosine at position 78 in the sequence of a protein. The Xsm represents a mutation at that position to a set of random amino acids which include the relatively small amino acids: alanine, cysteine, glycine, serine, threonine, and valine.

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

IMPROVING THE THERMOSTABILITY AND INCREASING THE SUBSTRATE RANGE OF OLD YELLOW ENZYME HOMOLOGS AND AMINOLEVULINIC ACID SYNTHASE


By

Robert Wilson Powell III

December 2016

Chair: Jon Dale Stewart Major: Chemistry

We examined the consequences of mutating the general acid at position Y196 in

OYE 1. We screened a library of OYE 1 Y196 site-saturation mutants against substrates

of interest and discovered that only the cysteine variant gave an interesting result. We

then crystalized this mutant to determine how a cysteine was acting as a general acid.

We discovered that it was definitely not properly positioned, and the identity of the

actual general acid remains unknown.

We also examined an OYE homolog, OYE 2.6. Previous protein engineering

done by our group, as well as revelations from the crystal structure, gave us new insight

into ways to target the active site for mutagenesis. By making a matrix of small amino

acids mutantations at two neighboring positions, we hoped to open up the active site

and make it more amenable to alpha substituted 6 membered rings. We found that a

third mutation on the opposite side of the active site would give further improvement.

We then set out to improve the thermostability of OYE 2.6 by directed evolution.

We chose positions within OYE 2.6 that had high B-values in the crystal structure for

mutagenesis. After successfully increasing the thermostability of OYE 2.6, we then set

21

out to prove our hypothesis by crystallizing the thermostable mutant and comparing the

B-values at the mutated position. We found that we had indeed lowered the B-factor

value at the position which we mutated.

We next examined OYE 3 as a biocatalyst. We made a library of OYE 3 W116

mutants and screened it against several substrates of interest. We then set out to solve

the crystal structure of OYE 3 so that we could more appropriately examine the

structure of our mutants in complex with our substrates.

Lastly, we waded in mutagenesis of a PLP-dependent enzyme, mALAS. We

made three libraries of mALAS targeting positions T148, I151, and R85. We hoped that

these positions would be malleable positions in the active site that would allow us to

expand the substrate range of mALAS.

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CHAPTER 1 PROBING POSITION Y196 IN OLD YELLOW ENZYME 1

Background

Isolation and Purification of OYE

Old Yellow Enzyme (OYE) was first isolated by Warburg and Christian from

brewers’ bottom yeast (Saccharomyces carlsbergensis; subsequently re-named

Saccharomyces pastorianus) in 19321 while studying the oxidation of glucose-6-

phosphate (G-6-P) to 6-phospho-D-glucono-1,5-lactone. Glucose-6-phosphate

dehydrogenase (G6PDH) oxidized G-6-P using NADP+, which produced NADPH. OYE

was responsible for oxidizing NADPH by reducing O2 to H2O2 (Figure 1-1).2 The enzyme

was termed a ‘gelbe ferment’, or a yellow ferment. In 1938, Warburg and Has

discovered a second type of ‘gelbe ferment,’ leading them to designate the original

‘gelbe ferment’ as the ‘old yellow enzyme’ (OYE)3 and the enzyme has been known by

this name ever since. In 1955, Theorell and Åkeson purified the enzyme from the lysate

of brewers’ bottom yeast using a series of organic solvents and then crystallized the

protein using ammonium sulfate precipitation.4

Since its original purification in 1955, OYE has been the subject of significant

study, particularly by the late Professor Vincent Massey. In 1968, Massey and Matthews

discovered that after purification, oxidized OYE formed a charge transfer complex with

an unidentified “green forming compound”.5 The “green forming compound” lost affinity

for OYE when the FMN cofactor was reduced by sodium dithionite and removing this

“green forming compound” by dialysis further improved purification of the enzyme.

Massey and Abramovitz would later use phenol as a ‘green forming compound’ to bind

to the oxidized enzyme on a phenol column as the basis for a very efficient affinity

23

purification method for OYE.6 After binding, OYE could be eluted by in situ sodium

dithionite reduction. This affinity purification strategy significantly improved the isolation

of OYE and the same phenol columns are still widely used today for purification of OYE

and its homologs.

Several OYE isoforms exist in brewers’ bottom yeast and these complicated

efforts to obtain diffraction-quality crystals. In 1991, Massey and Saito solved this

problem by isolating the gene encoding OYE from Saccharomyces carlbergensis and

cloning it into an E. coli expression plasmid (pET3b).7 Subsequently, two additional

OYE-encoding genes were cloned from Saccharomyces cerevisiae, leading the initial

gene to be designated as OYE 1. Massey and Stott cloned and overexpressed OYE 2

in 19938 and OYE 3 was cloned and overexpressed in 1995 by Massey and Niino.9

Catalytic cycle and substrates of OYE

During their experiments to purify OYE through crystallization in 1955, Theorell

and Åkeson discovered that OYE had a flavin mononucleotide (FMN) cofactor.4 Later

studies by Massey et al. investigated the oxidative half reaction of OYE. These spectral

studies of the OYE charge transfer complex established the catalytic cycle of OYE and

have opened the way for it to be used as a biocatalyst ever since (Figure 1-2).8,10,11

While NADPH appears to be the physiological reductant for OYE, the native partner for

flavin re-oxidation has never been discovered.

Several studies have been performed with non-native flavin re-oxidation

substrates for OYE 1, some of the first involving quinones tested by Massey and Stott

during spectral studies.8 This suggested that OYE 1 could reduce electron-deficient

alkenes at the expense of NADPH, which touched off an avalanche of interest in using

this enzyme for asymmetric chemical synthesis. A vast array of alkene substrates have

24

been tested with OYE 1. These include many α,β-unsaturated ketones, aldehydes,10–14

nitroalkenes,15–18 and even alkynes (Figure 1-3).19

Structure and Mechanism of OYE

Though OYE 1 was first purified and crystallized from lysate by Theorell and

Åkeson in 1955, its three-dimensional structure was not determined until 1994 when

Karplus and Fox successfully obtained diffraction quality crystals of OYE 1.4,20 The

overall structure of OYE 1 features an α/β-barrel (TIM barrel) with the active site buried

in the barrel along with the FMN cofactor. Within the active site, T37, G72, Q114, and

R243 form hydrogen bonds with the FMN and lock it into place (Figure 1-4). In 1998,

Massey and Brown used site directed mutagenesis and a series kinetic experiments to

establish the role of H191 and N194.21 These amino acids stabilize the oxyanion

intermediate prior to its protonation. In the same year, Massey and Kohli also examined

the role of Y196. Site directed mutagenesis was used to prepare a phenylalanine

substitution and the kinetic properties of both it as well as the wild type protein were

compared. Interestingly, the Y196F mutation had little effect on ligand binding; however,

the oxidative half reduction was nearly 6 orders of magnitude slower than the wild-

type.11 This study helped establish tyrosine as the general acid in the catalytic

mechanism of OYE 1 (Figure 1-5).

Our group has investigated the potential of OYE 1 as a biocatalyst.13,22,23 Much of

our work has been focused on preparing and testing various OYE 1 mutants for their

potential to accept synthetically interesting substrates. Amino acids mentioned above

are essential for FMN or substrate binding and / or catalysis and for this reason, were

not subjected to mutagenesis (Figure 1-7). On the other hand, we found that W116

25

accepted a wide variety of substitutions, some of which allowed for alternative (“flipped”)

substrate binding modes that led to opposite stereopreference (Figure 1-6).22–25

Project Overview

While Y196 was shown to be critical for the oxidation half-reaction and

associated with oxyanion protonation (Figure 1-5), we hypothesized that other residues

with even greater acidity might yield more efficient OYE 1 variants. We therefore

explored site-saturation mutagenesis of Y196 in OYE 1. A randomized library variants at

this position was created and screened against two alkene substrates (11 and 12 shown

in Figure 1-10). After finding catalytically active members in the library, plasmids were

sequenced and the final variant not already present was added. The complete Y196

site-saturation mutagenesis library was then screened against a broader panel of

substrates (Figures 1-8 through 1-10). These efforts revealed that the Y196C mutant

was catalytically active against a subset of the substrate collection. This was

reminiscent of the Y193C variant of Pichia stipitis OYE 2.6, a fascinating, if inconsistent,

mutant discovered in a parallel project by Adam Walton. To determine how a cysteine at

position 196 of OYE 1 could lead to substrate protonation, we determined the crystal

structure of this variant. We hoped that the information from this structure might shed

light on the analogous Y193C mutant of P. stipitis OYE 2.6 that has stubbornly resisted

all efforts at crystallization.

Results and Discussion

OYE 1 YI96 Site-Saturation Mutagenesis Library

We chose to prepare the site-saturation library en masse, deferring DNA

sequencing until it became clear that one or more active variants were actually present.

To this end, a randomized library of OYE 1 Y196 was made using a pair of primers with

26

a NNK mix at the codon for Y196 (N = any base, K = G / T). This base doping scheme

encompasses 32 different codons that includes all 20 amino acids plus one stop codon.

Using a variation of a cloning method reported by Zheng et al. that our group developed

for cloning NNK randomized libraries,26,27 PCR was performed and a pooled plasmid

sample obtained from transformants was sequenced. Using a method for evaluating

libraries developed in our lab by Sullivan and Walton,26 the plasmid mix was evaluated

for codon degeneracy. Sequencing of this pooled sample revealed a plasmid mix a

codon mix that gave a Q score of 0.85 (Figure 1-11), which implied that all 19 mutants

could be obtained with subsequent transformation into an E. coli overexpression strain.

A randomized library of 95 individual OYE 1 Y196 mutants (plus the wild-type control)

was assembled from transformants derived from the pooled plasmid sample. Each

variant was screened against 2-methyl-2-cyclohexen-1-one (substrate 12) to detect

catalytic activity. Plasmid DNAs from hits in this initial screen were sequenced to

determine the codon at position 196. The best variant was Y196C. This was fascinating

since an analogous mutant from P. stipitis OYE 2.6 (Y193C) also proved to be the

optimal substitution for the catalytic Tyr side-chain.28

These initial results were sufficiently promising to justify sequencing the

randomized library to determine whether all of the position 196 variants were actually

present and identify any that needed to be added to complete the set. These efforts

revealed 18 / 19 mutants, with only OYE 1 Y196W being absent. The original Q score

predicted that all 19 variants were present in the plasmid mix used to transform the E.

coli overexpression strain. Our obtaining 18 mutants was therefore very encouraging,

further underscoring the value and accuracy of our simple library quality control

27

determination. The missing OYE 1 Y196W mutant was made by standard methods,

then and the complete library was then used to screen other substrates (Figures 1-12

through 1-18). Unfortunately, none of the Y196 variants improved conversion for either

the Baylis-Hillman adducts (1 – 3) or either carvone enantiomer (7 and 8), with no

conversion for the former and minimal conversion for the latter. Also, no mutant gave

any activity against substrate 11, a screening substrate containing a 5-membered ring.

As expected from our initial screening efforts, we observed minor conversion for

substrate 12. The best variant (Y196C) gave racemic reduction product from 12 (wild-

type gives >98% ee (R)), although the conversion was only 20%.

Crystal Structure of OYE 1 YI96C

Given the difference in spatial locations between the acidic protons of a Tyr and

Cys residue, it was not clear how the Y196C mutant retained catalytic activity. We

therefore crystallized this OYE 1 variant in the hopes that the structure would provide

insight. 4-Hydroxybenzaldehyde (p-HBA) is an OYE inhibitor that binds strongly and can

provide information on the active site environment. We therefore soaked the mutant

crystals with p-HBA.

The OYE 1 Y196C mutant structure was near identical to the OYE 1 wt. structure

(1OYB) with a RMSD of 0.29 Å. The active site aligned nearly completely as well, with

the only difference being the Y196C mutation. Surprisingly, the structure revealed that

cysteine was not properly placed to participate directly in the alkene reduction step, as

its thiol moiety was directed away from the active site (Figure 1-19). In fact, the β-

carbon of the cysteine was 0.6 Å further than the tyrosine to the C2 of the p-HBA and

the sulfur was 5.0 Å further than the oxygen of the tyrosine. The thiol did not form a

disulfide bridge with a neighboring residue nor did it have an alternate oxidation state

28

like sulfenic (R-SOH), sulfinic (R-SO2H), or sulfonic acid (R-SO3H). This implies that a

general acid other than the Cys side-chain at position 196 acts as the general acid. The

actual proton source remains unknown, although it is intriguing that alkene 12 was

reduced to racemic product, consistent with solvent protonation after dissociation of the

enol(ate). On the other hand, this does not explain why the structurally analogous

carvone enantiomers were not accepted by the Y196C variant. While OYE is able to

tolerate the cysteine substitution, this change seriously impairs its function.

Experimental

General

Restriction endonucleases, Phusion Hot Start II High-Fidelity DNA Polymerase

and T4 DNA ligase were purchased from New England Biolabs. Primers were obtained

from Integrated DNA Technologies (IDT). All other reagents were obtained from

commercial suppliers and used as received. Plasmids were purified on small scales by

Wizard® minicolumns (Promega Life Sciences) and on large scales using CsCl density

gradient ultracentrifugation.29 DNA sequencing was carried out by the University of

Florida ICBR using capillary fluorescence methods using standard protocols. LB

medium contained 5 g/L Bacto-Yeast Extract, 10 g/L Bacto-Tryptone and 10 g/L NaCl.

ZY medium contained 5 g/L Bacto-Yeast Extract and 10 g/L Bacto-Tryptone. 50x5052

contained 25% glycerol, 2.5% glucose, and 10% a-lactose monohydrate. NPS x20

contained 66 g/L (NH4)2SO4, 136 g/L KH2PO4, and 142 g/L Na2HPO4.

29

Cloning

Construction of plasmids used to make the OYE 1 Y196 library

The plasmid used as a template to make the OYE 1 Y196 library was pET3b-

OYE (Appendix E Figure E-1), a pET3b derivative containing the gene for OYE 1.7

pET3b-OYE was originally a gift from Dr. Betty Jo Brown (University of Michigan).

Construction of mutants in the OYE 1 Y196X randomized library

All PCR samples were purified using Wizard® Plus SV Gel PCR Clean up kits by

Promega according to the manufacturer’s instructions. Samples were then incubated

overnight with two 0.5 μL aliquots of 20 U/μL DpnI at 37°C to remove the parent

template. The first portion of DpnI was added immediately after PCR clean up and the

second was added after 4 hours of digestion. After DpnI digestion, samples were

purified using Wizard® Plus SV Gel PCR Clean up kits by Promega.

Digested PCR samples were used to transform ElectroTen-Blue®

electrocompetent cells (ETB) using a Gene Pulser® from BioRad. Electroporation was

carried out with 4 μL of PCR sample and 50 μL of ETB cells using 2.5 kV.

Electroporated samples were incubated with 600 μL of SOC media at 37°C for 1 h.

Cells were then plated onto LB-amp agar plates and grown at 37°C for 36 h. The best

results were obtained from ETB daughter cells grown from the commercial stock on the

same day as the transformation would take place. Granddaughter cells provided fewer

transformants. Transformed cells were then pooled by rinsing the plate with a minimal

volume of LB and scraping with a rubber policeman. Plasmid DNA was extracted and

purified using Wizard® Plus minipreps DNA purification system by Promega according

to the manufacturer’s instructions. Purified, pooled plasmids were sequenced by ICBR

using Sanger sequencing. Raw electropherograms (chromat files) obtained from Sanger

30

sequencing were analyzed to estimate the samples degeneracy. Degeneracy could be

gauged for samples using a NNK primer mix (Figure 1-11) by the method developed by

Sullivan and Walton.26 This gave a Q-score of 0.85, suggesting that all position 196

variants were present in the library. A 4 μL aliquot of the purified, pooled plasmid was

used to transform overexpression strain E. coli BL21 (DE3) Gold cells using

electroporation (2.5 kV). Three transformants per possible codon would be required to

obtain a good representation for each possible codon in a pooled plasmid sample. For

this reason, 95 randomly-chosen colonies were used to seed 600 μL of LB-amp in a 96

well plate. Well H12 was reserved for the wild-type control. The plate was shaken

overnight at 37ºC to reach saturation. The library was stored by mixing 120 μL of each

culture with 30 μL of sterile 80% glycerol in a fresh 96 well plate. This gave a final

glycerol concentration of 15%, allowing the plate to be stored indefinitely at -80ºC.

Substrates

A list of substrates and products is shown if Figure 1-8 through 1-10.

2-(Hydroxymethyl)-cyclopent-2-enone (1)

2-(Hydroxymethyl)-cyclopent-2-enone was prepared in our lab by Bradford

Sullivan28 using the method developed by Kar and Argade.30 2-(Hydroxymethyl)-

cyclopent-2-enone can be detected during screening by GC-FID using a Beta Dex 225

column (0.25 mm × 30 m). The temperature program used began with an initial

temperature of 140°C for 10 min, followed by an increase at 20°C/min to a temperature

of 180°C at which the program remained for 5 min (GC method is listed as AZW2.Meth

in Appendix D). 2-(Hydroxymethyl)-cyclopent-2-enone eluted near 13.1 min. The

reduced products (S)- and (R)-6 eluted near 11.4 and 10.2 min, respectively.

31

2-(Hydroxymethyl)-cyclohex-2-enone (2)

2-(Hydroxymethyl)-cyclohex-2-enone was prepared in our lab by Bradford

Sullivan28 using the method developed by Rezgui and El Gaied.31 2-(Hydroxymethyl)-

cyclohex-2-enone was detected during screening by GC-FID using a Beta Dex 225

column (0.25 mm × 30 m). The temperature program used began with an initial



in Appendix D). 2-(Hydroxymethyl)-cyclohex-2-enone eluted near 13.1 min. The

reduced products (S)- and (R)-7 eluted near 10.2 and 10.8 min, respectively.

Methyl 2-(hydroxymethyl)acrylate (3)

Methyl 2-(hydroxymethyl)acrylate was prepared in our lab by Bradford Sullivan28

using the method developed by Turki et al.32 Methyl 2-(hydroxymethyl)acrylate was

detected during screening by GC-FID using a Beta Dex 225 column (0.25 mm × 30 m).

The temperature program began with an initial temperature of 100°C for 12 min,

followed by an increase at 20°C/min to a temperature of 180°C at which the program

remained for 5 min (GC method is listed as AZW3.Met in Appendix D). Methyl 2-

(hydroxymethyl)acrylate eluted near 11.8 min. The reduced products (S)- and (R)-7

eluted near 10.7 and 11.3 min, respectively.

(S)-(+)-Carvone (7)

(S)-(+)-Carvone was purchased from Sigma Aldrich and it can be detected by

GC-MS using a DB-17 column (0.25 mm × 30 m). The temperature program used

began with an initial temperature of 60°C for 2 min, followed by an increase at 10°C/min

to a temperature of 195°C at which the program remained for 10 min (GC method is

listed as JON.Meth in Appendix D). (S)-(+)-Carvone eluted near 12.5 min. A mixture of

32

reduced product isomers, (+)-Dihydrocarvone (Acros) was used as a standard to assign

the peaks for both cis- and trans-9 (11.7 and 11.3 min, respectively).

(R)-(-)-Carvone (8)

(R)-(-)-Carvone was purchased from Sigma Aldrich and it can be detected during

screening by GC-MS using a DB-17 column (0.25 mm × 30 m). The temperature

program began with an initial temperature of 60°C for 2 min, followed by an increase at

10°C/min to a temperature of 195°C at which the program remained for 10 min (GC

method is listed as JON.Meth in Appendix D). (R)-(-)-Carvone eluted near 12.5 min. A

mixture reduced product isomers, (+)-Dihydrocarvone (Acros) was used as a standard

to assign the peaks for both cis- and trans-10 (11.7. and 11.3 min, respectively).

2-Methyl-2-cyclopenten-1-one (11)

2-Methyl-2-cyclopenten-1-one was purchased from Sigma Aldrich. 2-methyl-2-

cyclopenten-1-one was detected during screening by GC-MS using a DB-17 column

(0.25 mm × 30 m). The temperature program began with an initial temperature of 60°C

for 2 min, followed by an increase at 10°C/min to a temperature of 195°C at which the

program remained for 10 min (GC method is listed as JON.Meth in Appendix D). 2-

methyl-cyclopenten-1-one eluted near 7.0 min, and the reduced product 13 eluted near

5.2 min.

2-Methyl-2-cyclohexen-1-one (12)

2-Methyl-2-cyclohexen-1-one was purchased from Sigma Aldrich. 2-methyl-2-

cyclohexen-1-one was detected during screening by GC-MS using a DB-17 column

(0.25 mm × 30 m). The temperature program began with an initial temperature of 60°C

for 2 min, followed by an increase at 10°C/min to a temperature of 195°C at which the

program remained for 10 min (GC method is listed as JON.Meth in Appendix D). 2-

33

methyl-2-cyclohexen-1-one eluted near 8.5 min and the reduced product 14 eluted near

7.2 min.

Screening Assay

E. coli BL21 (DE3) Gold cells harboring plasmids containing OYE 1 Y196

mutants of interest were grown in a 96 well plate containing 600 μL of LB-amp. Cells

were grown at 37°C with 250 rpm of agitation overnight. The saturated cultures were

then used to inoculate a larger 2 mL square bottom 96 well plate. This larger square

bottom plate contained 600 μL of an auto-induction media. The auto-induction medium

contained a mix of ZY media, 50x5052, 20x NPS, and 200 μg/mL ampicillin.33 Cells

were induced in an aeration case developed at the University of Florida by the machine

shop in the Department of Chemistry. Induction occurred at 37°C with 350 rpm of

agitation overnight. The increased agitation is required for induction to occur. Induced

cells were then separated from the auto-induction medium by centrifugation. The

supernatant was removed and the induced, pelleted cells were resuspended in 600 μL

of reaction mixture, which contained 50 mM KPi, 100 mM glucose and 15 mM alkene

substrate, pH 7.0. Reactions were shaken at 250 rpm overnight at room temperature

before quenching by adding 500 μL of ethyl acetate. The organic phase was separated

by centrifugation and analyzed by GC.

Protein Purification and Crystallogenesis of Y196C OYE 1

The Y196C OYE 1 mutant was purified by the same methods used previously for

wild-type and mutant OYE 1,34 which is a modification of the procedure originally

developed by Massey.6 E. coli BL21 (DE3) Gold cells harboring pET3b-OYE 1 Y196C

were grown at 37°C in a 4 L New Brunswick Scientific M19 fermenter containing LB-

amp. Cells were grown in the fermenter with 600 rpm of agitation for 2 h to achieve mid

34

log phase. Protein overproduction was induced by adding IPTG and glucose to final

concentrations of 0.4 mM and 100 mM, respectively. The culture was grown at 30°C

with 600 rpm of agitation for 4 h. The culture was then chilled at 4°C for 30 min before

centrifugation at 5,000 × g. The wet cell pellet was then resuspended in 100 mM Tris-Cl

buffer containing 10 μM phenylmethane sulfonyl fluoride (PMSF) at pH 8.0. Cells were

then lysed under 12,000 psi with the aid of a French press. Cell extract was centrifuged

at 18,000 × g for 1 h to remove insoluble debris. Nucleotides were precipitated by

adding protamine sulfate to a final concentration of 1 mg/mL and stirring at 4°C for 20

min. The supernatant was separated by centrifugation at 18,000 × g for 20 min. Protein

was precipitated out of solution by adding solid ammonium sulfate in 5 portions every 5

min to achieve a final concentration of 78% saturation. The protein precipitate was then

separated by centrifugation at 18,000 × g for 1 h.

Purification of OYE 1 by an N-(4-hydroxybenzoyl) aminohexyl agarose affinity

column requires that the active site be emptied of any bound ligand that would interfere

with binding to the phenol moiety of the column matrix. This was accomplished by

successive buffer exchanges during dialysis. The ammonium sulfate pellet obtained

from the salt cut was resuspended in 100 mM Tris-Cl, 100 mM (NH4)2SO4, 10 μM

PMSF, pH 8.0. This was dialyzed against 1 L of 100 mM Tris-Cl, 100 mM (NH4)2SO4, 10

μM PMSF, pH 8.0 overnight at 4ºC. The sample was then dialyzed against 1 L of the

same buffer containing 10 mM sodium dithionite for 2 h at 4ºC. After 2 h, the buffer was

exchanged for a fresh 1 L of buffer containing 10 mM sodium dithionite and dialysis

continued for 2 h. The sample was then transferred to a fresh 1 L of buffer without

dithionite and dialyzed for 2 h, after which, the buffer was exchanged with a final 1 L of

35

fresh buffer and dialyzed overnight. The final sample was then centrifuged at 18,000 × g

for 30 min to remove any insoluble debris accumulated during dialysis.

Dialyzed protein samples were loaded onto the affinity column in 10 mL portions.

The affinity column was equilibrated with 100 mM Tris-Cl, 100 mM (NH4)2SO4, 10 μM

PMSF, pH 8.0. Binding of OYE 1 turned the column green. After washing with 30 mL of

starting buffer, the desired protein was eluted by washing with 100 mM Tris-Cl, 100 mM

(NH4)2SO4, 10 μM PMSF, 4 mM sodium dithionite, pH 8.0. OYE 1 Y196C was then

further purified by gel filtration with a Superdex 200 column (Pharmacia) using 50 mM

Tris-Cl, 50 mM NaCl, pH 7.5. Pooled fractions containing the desired protein were then

concentrated by ultrafiltration using an Amicon centrifugation tube to a final

concentration of 20 mg/mL. Protein concentration was determined by absorbance at

280 nm using an extinction coefficient (ε) and molecular weight (MW) estimated by

protparam (OYE 1 Y196C had an ε of 70,820 M-1cm-1 with a MW of 44,954 Da).35

Crystals were grown using the published conditions discovered by Fox and

Karplus.20 Wells contained 6 μL of 20 mg/mL protein in 50 mM Tris-Cl, 50 mM NaCl, pH

7.5 and used hanging drop vapor diffusion. The crystallization solution contained 35%

(v/v) PEG 400, 100 mM Na HEPES, 200 mM MgCl2, pH 8.3. The best crystals obtained

were obtained after 10 days at 6°C. After crystallization, crystals were mounted in

appropriate loops and soaked with p-HBA before being flash cooled in liquid nitrogen

and sent for data collection. No cryoprotectant was used.

The Data Collection and Crystal Structure of OYE 1 Y196 Mutants

The best crystals diffracted to a maximum usable resolution of 1.36Å using the

X6A beamline at Brookhaven National Laboratory. The unit cell measured was 141.1

141.1 42.8 Å 90 90 90 and the crystals belonged to space group P 43 21 2. The

36

asymmetric unit contained 1 molecule with a solvent content of 48.05% and a Matthew’s

coefficient of 2.37 Å3/Dal.36

Reflection data were processed using the iMOSFLM program from the CCP4

program suite to a resolution of 1.36Å.37 Phases were obtained using the Phaser-MR

utility of the PHENIX program suite by molecular replacement using a modification of S.

pastorianus OYE 1 (PDB code 1OYB) as the search model.38 All ligands and water

molecules were removed prior to molecular replacement. Inspection of the model

showed one OYE 1 chain present in the asymmetric unit. The best solution for the

space group was determined to be P 43 21 2. The initially calculated 2Fo-Fc and Fo-Fc

maps showed electron density patterns that could be easily identified as the FMN

cofactor. The FMN cofactor was modeled into the structure. Initial refinement using the

xyz coordinates, B-factors, real-space, and occupancies refinement strategy features in

PHENIX refine as well as continued cycles of model building with the aid of the structure

validation tools in COOT produced a model with an Rfree of 0.1824.39 Continual

iterations of using the structural validation tools in COOT and PHENIX.refine produced

a model with an Rfree of 0.1698. At this point the p-HBA was modeled into the active site

and subsequent rounds of model building with COOT and refinement produced an Rfree

of 0.1554.

Conclusions

The Y196 mutants of OYE 1 showed no improvement of product range for the

enzyme. However, the observation that Y196 could be successfully replaced by Cys is

intriguing since the crystal structure of the mutant with bound p-HBA showed that the

cysteine residue was not appropriately positioned to act as a general acid. Our current

37

hypothesis is that solvent supplies the proton to the enol(ate) formed by enone

reduction, possibly after dissociation from the active site.

These OYE 1 structural data may also provide guidance for the analogous

Y193C mutant of P. stipitis OYE 2.6. The latter could not be crystallized because the

protein could not be purified with reproducible properties. Like OYE 1, Cys was the sole

functional replacement for Tyr in P. stipitis OYE 2.6. Whether the structure and catalytic

mechanism of this mutant resembles its OYE 1 counterpart remains to be determined.

38

Figure 1-1. The catalytic cycle of G6PDH investigated by Warburg and Christian.1,28

Figure 1-2. The catalytic cycle of OYE 1 established by Massey and Vas.10,40

39

Stott et al. (1993)8

Vaz et al. (1995)10

Figure 1-3. List of OYE 1 substrates from the literature.

40

Vaz et al. (1995)10

Figure 1-3. (Continued).

41

Vaz et al. (1995)10

Kohli et al. (1998)11

Meah et al. (2000)15


42

Meah et al. (2001)16

Williams et al. (2004)17

Swiderska et al. (October 2006)12


43

Swiderska et al. (December 2006)18

Mueller et al. (March 2007) 19 Bougioukou et al. (2008)13

Mueller et al. (September 2007)41


44

Hall et al. (2008)42

Padhi et al. (2009)23

Winkler et al. (2010)43


45

Stueckler et al. (September 2010)14

Stueckler et al. (October 2010)44

Brenna et al. (June 2011)45


46

Brenna et al. (July 2011)46

Brenna et al. (December 2011)47

Brenna et al. (2012)48

Durchschein et al. (2012)49


47

Tasnadi et al. (March 2012)50

Tasnadi et al. (June 2012)51


48


Brenna et al. (January 2014)53


49


Turrini et al. (2015)55


50

Figure 1-4. FMN Diagram displaying the FMN (gold) environment in the active site of OYE 1. OYE 1 (green) uses hydrogen bonding partners to lock FMN in place within the active site (T37, G72, Q114, and R243). OYE 1 also uses the hydrophobic bonding partners beneath to FMN shown with blue circles (P35, L36, and I351) to lock it into position.

Figure 1-5. Catalytic mechanism of OYE 1. Mechanism for the reduction of a bound α-unsaturated carbonyl substrate (black) by a reduced FMN (gold) in the active site of OYE 1 (green). OYE 1 uses positions H191 and N194 as hydrogen bonding partners to lock the carbonyl into position.

51

Figure 1-6. OYE substrate binding modes. In both binding modes, cyclohexenone docks in the active site above and parallel to the plane of the reduced FMN. The carbonyl oxygen forms hydrogen bonds with the residues N194 and H191. The hydride from N5 is transferred to the electron deficient β-carbon. Y196 acts as a general acid to protonate the resulting enolate (see figure 1-5) at the α-carbon. The “flipped” conformation requires a shift in the angle of hydride and proton transfer and is sterically crowded by the presence of Trp116. The stereochemistry of each binding mode product is indicated with the protic hydrogen (green) and the hydride hydrogen (gold) below each scheme. For a prochiral substrate the two binding modes determine product stereochemistry.28

52

Figure 1-7. OYE 1 active site diagram. This diagram shows the areas of the OYE 1

active site. The orange section contains the “east side” of the active site which will be discussed more in chapter 2. The magenta section contains other active site positions which form a gate with the positions on loop 6. Loop 6 is the mobile part of this gate and opens up to allow NADPH to enter the active site and reduce oxidized FMN. The green section includes position on loop 6 which will be of discussed more in chapter 4. The Blue segment contains the “west side” of the active site which will be discussed more in Chapter 2. The yellow segment contains positions that interact with the bound substrate (N194 and H191) and position Y196 which acts as a general acid in OYE 1 catalysis.

53

Figure 1-8. List of Baylis-Hillman substrates screened by OYE 1 Y196 library.

54

Figure 1-9. List of carvone substrates screened by OYE 1 Y196 library.

55

Figure 1-10. List of screening substrates screened by OYE 1 Y196 library.

56

Figure 1-11. Calculations for obtaining a Qscore of a pooled plasmid mix from a NNK primer mix using data from a theoretical sequencing electropherogram. This figure shows a theoretical electropherogram with theoretical peak heights. a) The fraction of each peak height at a position over all peak heights at that position is shown on the left side of each peak. Peaks correspond to: blue/thymine, orange/guanine, green/adenine, purple/cytosine, and the dashed black represents perfect degeneracy. b) The peak fraction of each base is used to estimate the amount of codons containing that base at that position. c) The sum of those estimates are used to obtain a Q value (QN or QK) for that position. d) The sum of the weighted QN and QK values is used to calculate the Qscore for the pooled mix. The Qscore approaches perfect degeneracy (equal amounts of bases at each position) as it approaches 1.0.26

57

Figure 1-12. OYE 1 Y196 library screening results for 1. No conversion was observed for any mutants. OYE wt. results are consistent with previous findings.22


58


Figure 1-15. OYE 1 Y196 library screening results for 7. OYE wt. results for (S)-(+)-carvone are consistent with previous findings.24

59

Figure 1-16. OYE 1 Y196 library screening results for 8. OYE wt. results are consistent with previous findings.24

Figure 1-17. OYE 1 Y196 library screening results for 11. The stereochemistry of products for substrate 11 was not evaluated during screening.

60

Figure 1-18. OYE 1 Y196 library screening results for 12. The stereochemistry of products for substrate 12 was not evaluated during screening. The stereochemistry of OYE 1 Y196C was evaluated after initial screening by GC-FID using a Beta Dex 225 column (0.25 mm x 30 m) using AZW2.Meth (Appendix D). Peaks were never assigned.

Figure 1-19. Alignment of OYE 1 wt. and OYE 1 Y196C. This figure shows OYE 1 wt. (green) aligned to and OYE 1 Y196C (orange) (PDB ID 1OYB) with FMN cofactor (yellow). Both structures have p-HBA bound in the active site.20

61

CHAPTER 2

IMPROVING THE PRODUCT RANGE OF OYE 1 AND OYE 2.6 THROUGH PROTEIN ENGINEERING

Background

Mutagenesis of the East Side of the Active Site in OYE

Given the potential of alkene reductases for making products with high

enantiomeric excess, we were eager to explore this class of enzymes for use as a

biocatalysts. Long before we waded into an expansive mutagenesis projects which are

frequent in this group now, we wanted to probe a set of alkene reductases against a

series of substrates. To this end, our lab made an alkene reductase library with 16

alkene reductases.56 This collection of alkene reductases was assembled by Despina

Bougioukou and was screened against a series of substrates of interest.56 Table 2-1

lists all the fully sequenced alkene reductase libraries made by the Stewart group.

One enzyme in that library that was of interest to our group was Old Yellow

Enzyme (OYE 1) from Saccharomyces pastorianus. Using the crystal structure of OYE

1 solved by Fox and Karplus,20 our group identified a position in the active site that we

believed might affect the orientation of substrate binding and could influence the type of

products we could obtain. Position W116 is located on the east side of the OYE 1 active

site and the tryptophan at this position is in close proximity to groups that extend off the

alpha carbon of any bound carbonyl substrate. We believed that substitution of this

bulky tryptophan for a different residue may allow substrates to bind in an alternate

“flipped binding mode”. Figure 2-1 shows the flipped binding mode in the OYE 1 active

site. Binding in this mode would give the alternate enantiomer at the alpha carbon

following catalysis. To pursue this idea, in 2009 Despina Bougioukou and Santosh

http://www.uniprot.org/taxonomy/27292

62

Padhi used site saturation mutagenesis at position W116 of OYE 1 to discover new

mutants that would allow flipped binding.23 Our group was very interested in a set of

Baylis-Hillman substrates (substrates 1-3) as well as a pair of carvone substrates

(substrates 7 & 8) (Figure 2-2). Bougioukou and Padhi screened the carvone substrates

against the OYE 1 W116 mutants. The blind screening done with these mutants

identified functional mutations including OYE 1 W116F, W116I, W116L, W116M and

W116Y. It was also discovered that OYE 1 W116I would flip (S)-(+)-carvone and give

the trans product, trans-9 (diasteriomeric excess (de) = 88% in favor of trans and

conversion was 98%). OYE 1 wild type (wt) however, gave the cis product (cis-9) for

(S)-(+)-carvone (de = 93% in favor of cis and conversion was 48%). These findings

were significant enough to warrant making a full degenerate library containing all 20

residues substituted at position W116. In 2011, Adam Walton and coworkers screened

the Baylis-Hillman substrates against this OYE 1 W116 library.22 They found that some

of these mutants would allow flipped binding for 2-(hydroxymethyl)-cyclopent-2-enone

(substrate 1) and methyl 2-(hydroxymethyl)acrylate (substrate 3). In 2013, Yuri Pompeu

and Bradford Sullivan screened the carvone substrates against the completed OYE 1

W116 mutants.24 They identified new mutants that could provide the alternate trans

product (trans-9) for (S)-(+)-carvone (OYE 1 W116A, W116C, W116E, W116G, W116I,

W116M, W116N, W116Q, W116S, W116T, and W116V) as well as the alternate cis

product (cis-10) for (R)-(-)-carvone OYE 1 W116A, and W116V). Furthermore, the

crystallographic studies done by Pompeu and Sullivan showed (S)-(+)-carvone bound in

the flipped binding mode within the active site of the OYE 1 W116I mutant. Figure 2-3

shows the active site of OYE 1 W116I with (S)-(+)-carvone bound.24 And as such, this

63

work provided firm evidence for the idea that position W116 played a role in

discriminating substrates during binding and that mutating this position would make the

active site more amenable to a flipped binding mode which could alter the

stereochemistry of the products.

Another OYE homolog that our group investigated was OYE 2.6 from Pichia

stipitis. OYE 2.6 has an isoleucine at position I113 which is the analogous position to

W116 in OYE 1. What is fascinating about OYE 2.6 is that it gives the same cis product

(cis-9) as OYE 1 wt for (S)-(+)-carvone. This is noteworthy because when these two

homologs have the same residue at an analogous position they give different products.

Because of this, our group began focusing on OYE 2.6 as a biocataylst.34,57,58 In one of

our most ambitious mutagenesis projects to date, our group extensively mutated several

positions in the OYE 2.6 active site to explore its potential for engineered

biocatalysis.34,57,58 The approach our group used during this project was a technique

developed by Manfred Reetz called iterative saturation mutagenesis (ISM).59 ISM is a

progressive protein engineering strategy that targets several positions of interest over a

series of mutagenic rounds. The best mutant identified for each position is then used as

an “anchor” for a second round of mutagenesis where a different position is

randomized. Successive rounds are carried out with the best mutant at each position

being added to the next round of randomization. The goal of this strategy is to hone in

on multi-mutated variants with exceptional properties. Targeting positions around the

OYE 2.6 active site, our lab made several 1st, 2nd, and 3rd generation libraries.57 Figure

2-4 has a diagram showing all the residues in the OYE 2.6 active site. The ISM

experiments were concluded after variants were discovered that would allow the flipped

64

binding mode and give the (R)- product with high enantiomeric excess (ee) for two of

the three Baylis-Hillman substrates. These variants worked for both substrate 3 and

substrate 1.57 A 2nd generation library uncovered an OYE 2.6 Y78W / F247A double

mutant that gave the best results for the 2-(hydroxymethyl)-cyclohex-2-enone (substrate

2). Table 2-2 summarizes the best variants of OYE 1 and OYE 2.6 for obtaining de and

ee for carvone and Baylis-Hillman substrates. The best result however was a

conversion 43% and an ee of 37% (S), which is not ideal since the product of interest is

the R enantiomer ((R)-5). As such, a good solution for obtaining the (R)-5 product from

2 was not discovered during the ISM project.

Project Summary

Though an enzyme that would exclusively make the (R)-5 product from 2 was

never discovered, we did find a set of promising positions that gave us a good idea on

how to move forward. Since OYE 1 wt only gives 10% conversion for 2 we decided to

focus our efforts on engineering OYE 2.6 which gives nearly 100% conversion. We

believed that the answer would be found at the east side of the active site. Molecular

modeling shows that the alternate flipped binding mode of 2 would crash into the east

side residues in OYE 2.6. Figure 2-7 shows OYE 2.6 with a 6-membered ring modeled

into the active site. The best double mutant combination identified during the ISM

project with a pair of east side mutants, was OYE 2.6 Y78W / I113C. This double

mutant includes two positions that are located on the east side of the active site and

their residues extend into the space where any substrate that attempts a flipped binding

to OYE 2.6 would occupy. Figure 2-6 shows the crystal structure of OYE 2.6 Y78W /

I113C with malonate bound into active site. The best triple mutant discovered for

obtaining the (R)-5 product from 2 was OYE 2.6 Y78W / I113C / F247A which includes

65

the two mutants at positions on the east side of the OYE 2.6 active site of interest as

well as one position on the west side.57 Given the results of these variants, we wanted

to further investigate these three positions.

The main aim of this project was to find a combination of mutations that would

improve the yield for the (R)-5 product for OYE 2.6 and in doing so, hopefully discover

new biocatalysts that could be used on other future substrates. In this project we

examine what would happen if we compacted the large obstructive steric bulk on the

east side of the active site in OYE 2.6. We began by making a randomized matrix library

of the two positions on the east side of the active site that had the most notable effect

on enantiomeric excess during the ISM experiments, positions Y78 and I113. These

positions would be replaced with a pair of smaller residues; alanine, cysteine, glycine,

serine, threonine, and valine. We then assembled a library with presequenced double

mutants at those positions. Since the best double and triple mutant variants contained

mutations at position F247, that was another position we wanted to target during this

project. Since we believe that position Y78 and I113 interact with each other, it was

important to find the right combinations at these two positions before looking at the

F247 position. After anchoring off the best double mutants discovered during the

mutagenesis of the east side of active site, a set of 2nd round libraries were made

targeting the F247 position located on the west side of the active site.


OYE 2.6 Y78Xsm / I113Xsm Randomized Library

Initial cloning simultaneously targeted two positions located on the east side of

the OYE 2.6 active site, a tyrosine at position Y78 and an isoleucine at positon I113.

The aim of this strategy was to replace these two residues with a pair of small amino

66

acids which would take up less space and allow the alternate flipped binding mode for 2

which would in turn produce more (R)-5 product. The smaller pair of residues would

include a combination of either alanine, cysteine, glycine, serine, threonine or valine.

Given the substantial amount of cloning that had already been done on this enzyme

(fourteen 1st generation, nine 2nd generation, and two 3rd generation libraries)28,57 it was

preferable to survey the active site with as minimal effort as possible. To minimize the

laborious task of cloning a matrix of double mutants, a randomized cloning approach

was chosen to assemble the first library. A KST random primer mix contains an equal

number of codons for alanine, cysteine, glycine, and serine. Anchoring off a pBS2

vector, a pET derivative with GST-OYE 2.6 fusion protein (Appendix E, Figure E-2), and

primers using a KST mix at position Y78 would provide a mix of mutants containing

many of the single mutations needed. Anchoring off of this random pBS2-OYE 2.6

Y78XKST plasmid mix, primers using a KST mix at position I113 provided a random mix

containing up to 16 of the double mutations of interest. Anchoring off OYE 2.6 Y78T,

and OYE 2.6 Y78V with OYE 2.6 I113XKST primers as well as anchoring off OYE 2.6

I113T and OYE 2.6 I113V with OYE 2.6 Y78XKST primers provided us with four random

mixes containing up to 16 more double mutations of interest. The final four threonine-

valine double mutants had to be made individually. In this way the number of PCR

reactions, and the subsequent cloning steps, was reduced. Mutants obtained from both

the pooled KST randomized PCR cloning, and the completed set of valine and

threonine double mutants were used to assemble a random library containing small

residues at both the Y78 and the I113 position.

67

The three Baylis-Hillman substrates were used to screen the mutants in the OYE

2.6 Y78Xsm / I113Xsm randomized library. The positions that gave the most notable

results were then sequenced to determine the mutations present. By sequencing the

mutants after screening, we save time and effort by not sequencing numerous less

successful mutants. The majority of successful mutants were double mutants that were

successfully cloned. However, in a few cases promising positions were revealed to

contain concatomeric repeats of primer inserts in the sequence at the Y78 position.

Figure 2-8 shows the alignment of OYE 2.6 sequence with a sample containing portions

of primer inserts in the sequence. No concatomeric repeats of the primer was observed

in samples in which the I113 was targeted for cloning.

Positions in the OYE 2.6 Y78Xsm / I113Xsm randomized library that produced the

(R)-5 product were sequenced. Sequencing revealed that a cysteine mutation at

position I113 was present in most of the successful mutants. These substrates had

been extensively screened against an OYE 2.6 I113C single mutant and an OYE 2.6

Y78W / I113C double mutant in previous work.57 In fact, during the ISM experiments

anchoring off OYE 2.6 Y78W in which position I113 was randomized, it was revealed

that OYE 2.6 Y78W / I113C was the best double mutant in that library. OYE 2.6 I113C

and OYE 2.6 Y78W / I113C gave 100% and 98% conversion and 81% and 60% ee (S)

for the (S)-5 product of 2 respectively (Figure 2-9). However, the OYE 2.6 Y78Xsm /

I113C mutants had performed better than the single OYE 2.6 I113 and the OYE 2.6 Y78

/ I113 double mutant variants from the ISM experiments. Since these experiments,

which included a small amino acid mutation at position Y78, provided improved results

for obtaining the (R)-5 product from 2, we decided to fully explore the potential of double

68

mutants at these two positions. Thus, a complete library was assembled containing a

full matrix of all 36 double mutants.

OYE 2.6 Y78Xsm / I113Xsm Presequenced Library

Given the promising results of some of the mutants in the OYE 2.6 Y78Xsm /

I113Xsm randomized library, efforts were made to make all the small amino acid double

mutants of OYE 2.6 at positions Y78 and I113. Though the best results obtained from

screening the OYE 2.6 Y78Xsm / I113Xsm randomized library all contained the I113C

mutation, it was a concern that if we solely focused on the OYE 2.6 I113C double

mutants we might miss variants with remarkable results. Therefore, all 36 double

mutants would be made and screened. Substrate 2 was screened against the mutants

of the completed OYE 2.6 Y78Xsm / I113Xsm presequenced. Most of the mutants present

provided excellent conversion for 2. Figure 2-10 shows the results of screening the OYE

2.6 Y78Xsm / I113Xsm presequenced library against 2. However, the only set of mutants

that gave any (R)-5 product were the I113C mutants. Every OYE 2.6 I113C mutant gave

at least some (R)-5 product. Though no mutation at position Y78 stood out as the clear

choice for obtaining better yields of (R)-5, I113C was clearly the best substitution at that

position. At this point we felt there was not much more that could be done with solely

looking at these two positions. Moving forward, we chose to target a position that

worked well in combination with other double mutants made on the east side of the

active site, position F247.

OYE 2.6 Y78Xsm / I113C / F247X Randomized Libraries

We then investigated the west side of the OYE 2.6 active site, anchoring off the

best the double mutants discovered while screening the presequenced OYE 2.6 Y78Xsm

/ I113Xsm library. Previous mutagenesis projects57 revealed that a set of OYE 2.6 Y78W

69

/ I113C / F247A and F247H triple mutants would give good results for 2. Also, double

mutants that included a F247 mutation were in fact the best double mutants discovered

in the ISM project for obtaining (R)-5. Anchoring off of OYE 2.6 Y78A, Y78C, Y78G,

Y78S, Y78T, and Y78V / I113C, a set of 6 libraries were made in which position F247

was randomized with a set of primers containing a NNK mix of bases at the codon for

F247. A NNK mix contains 32 codons which include at least one codon for every

residue. This is the approach our group used during the ISM experiments to make

mutagenic libraries. Pooled samples of transformants were sequenced to access their

degeneracy using an evaluation method developed in our group during the ISM

experiments.26 Samples with sufficient degeneracy were further cloned into expression

strains and assembled into a randomized library. Table 2-3 contains all the Q scores

and the estimated number of amino acids obtainable from transformation with that

plasmid mix. Substrate 2 was used to screen the six OYE 2.6 Y78Xsm / I113C / F247X

libraries (Figure 2-11 through 2-16 shows the results of triple mutant screenings).

Unfortunately, few of the triple mutant variants gave any significant improvement over

the double-mutant anchor used to make the library. The six best results were

sequenced and not surprisingly four were the double mutant controls. The only two

triple mutants discovered during sequencing were OYE 2.6 Y78C / I113C / F247H and

F247W. With near 100% conversion and racemic ee, these mutations are the best

results discovered during this project and best variants of OYE 2.6 for obtaining (R)-5

from substrate 2 (Figure 2-9).

70

Experimental

General



from Integrated DNA Technologies (IDT). All other reagents were obtained from

commercial suppliers and used as received. Plasmids were purified on small scales by

Wizard® minicolumns (Promega Life Sciences) and on large scales using CsCl density

gradient ultracentrifugation.29 DNA sequencing was carried out by the University of

Florida ICBR using capillary fluorescence methods using standard protocols. LB

medium contained 5 g/L Bacto-Yeast Extract, 10 g/L Bacto-Tryptone and 10 g/L NaCl.

ZY medium contained 5 g/L Bacto-Yeast Extract and 10 g/L Bacto-Tryptone. 50x5052

contained 25% glycerol, 2.5% glucose, and 10% a-lactose monohydrate. NPS x20

contained 66 g/L (NH4)2SO4, 136 g/L KH2PO4, and 142 g/L Na2HPO4.

Cloning

Construction of plasmids used to make the OYE 2.6

The plasmid used as a template to make the OYE 2.6 libraries was pBS2, a

pET21a (+) derivative containing the gene for OYE 2.6-GST fusion protein. It was made

by the combined efforts of Despina Bougioukou and Bradford Sullivan (Appendix E,

figure E-2).34

Construction of the mutants in the OYE 2.6 Y78Xsm / I113Xsm randomized library

Templates used to make the mutants in the OYE 2.6 Y78Xsm / I113Xsm

randomized library were pBS2 derivatives obtained from an OYE 2.6 I113

presequenced library assembled by Adam Walton.34,57 Primers containing a KST mix of

nucleic acids at the OYE 2.6 Y78 position were used to make the pBS2-OYE 2.6

71

Y78XKST (OYE 2.6 Y78A, Y78C, Y78G, and Y78S) single mutant plasmid mix. Using the

pBS2-OYE 2.6 Y78XKST plasmid mix as a template, primers containing a KST mix of

nucleic acids at the OYE 2.6 I113 position were used to make the remaining OYE 2.6

Y78XKST / I113XKST double mutant plasmid mix. Primers containing a codon to give the

OYE 2.6 Y78T and Y78V mutations were used to make the four OYE 2.6 Y78T and

Y78V / I113T and I113V double mutants. Primers containing a KST mix of nucleic acids

at the OYE 2.6 Y78 and I113 positions were used with the appropriate pBS2 double

mutant to make the OYE 2.6 Y78XKST / I113T, OYE 2.6 Y78XKST / I113V, OYE 2.6 Y78T

/ I113XKST, and OYE 2.6 Y78V / I113XKST double mutant mixes. All primers used in this

chapter are listed in Appendix A, Table A-2. PCR was performed using 0.5 µL of 18 ng/

µL template, 5 µL of both 5 mM forward and reverse mutagenic primers, 1 µL of 10 mM

dNTP mix, 10 µL of 5X HF Phusion® Hot start buffer, 28 µL of sterile water, and 0.5 µL

of 2 U/µL Phusion® Hot Start II DNA Polymerase for a total reaction volume of 50 µL.

PCR was performed using a MJ Mini® thermocycler from BioRad. PCR samples were

run with an initial denaturation step at 98°C for 30 s, then a subsequent 25 cycles of

denaturation at 98°C for 10 s, annealing at 64°C for 30 s, and an extension step at 72°C

for 3 min 30 s, after which the reactions were completed with a final extension step at

72°C for 7 min 30 s.

All PCR samples were cleaned using Wizard® Plus SV Gel PCR Clean up kits by

Promega, using the manufacturer instructions. Samples were then incubated overnight

with two doses of 0.5 µL of 20 U/µL DpnI at 37°C to remove the parent template. DpnI is

a nuclease that targets hemi methylated DNA and as such is ideal for removing the

superfluous template. The first dose of DpnI was added immediately after PCR clean up

72

and the second was added after 4 hours of digestion. After DpnI digestion, samples

were cleaned using Wizard® Plus SV Gel PCR Clean up kits by Promega, using the

manufacturer instructions.

Following the PCR work up, PCR samples were transformed by electroporation

into ElectroTen-Blue® electrocompetent cells (ETB) using a Gene Pulser® from BioRad.

Electroporation was carried out with 4 µL of PCR sample and 50 µL of ETB cells under

2.5 kV. Electroporated samples were incubated in 600 µL of SOC medium at 37°C for 1

h. Cells were then plated onto LB-amp agar medium and grown at 37°C for 36 h. The

best results were obtained from ETB daughter cells grown from the commercial stock

on the same day as the transformation would take place. Granddaughter cells provided

fewer transformants. Transformant cells were then pooled and plasmid DNA extraction

was performed with Wizard® Plus minipreps DNA purification system by Promega, using

the manufacturer’s instructions. Pooled plasmid samples were then sequenced by ICBR

using Sanger sequencing. Electropherograms obtained from Sanger sequencing were

measured to estimate the samples degeneracy. Degeneracy could be gauged for

samples using a KST primer mix (figure 2-17) by the method developed by Adam

Walton and Bradford Sullivan.26 Pooled plasmid samples with sufficient degeneracy

were used to transform an expression strain, E. coli BL21 (DE3) Gold cells.

Transformation was done using electroporation under 2.5 kV with 4 µL of 10 ng/µL of

pooled plasmid with 80 µL of E. coli BL21 (DE3) Gold electrocompetent cells.

Electroporated samples were incubated in 600 µL of SOC media at 37°C for 45 min.

Cells were then plated onto LB-amp agar medium and grown at 37°C overnight.

Transformants were then assembled into two 96 well plates. Three transformants per

73

possible codon would be required to obtain a good representation for each possible

codon in a pooled plasmid sample. Therefore, twelve transformants were taken from

each transformation of PCR samples using KST primer mix to accommodate the four

codons possible in a single KST codon mix, and 48 transformants were taken from the

transformation of the PCR sample using a double KST primer mix. Transformants were

grown in 600 µL of LB-amp in a 96 well plate overnight to reach saturation. The library

was completed with the transfer of 120 µL of saturated cultures into a new 96 well plate

containing 30 µL of 80% glycerol which brought the final concentration of glycerol to

15%.

Construction of the mutants in the OYE 2.6 Y78Xsm-I113Xsm presequenced library

The primers used for constructing a presequenced OYE 2.6 Y78Xsm-I113Xsm

library were from IDT (Appendix A, Table A-2). The plasmids were pBS2 derivatives

obtained either from the OYE 2.6 Y78Xsm-I113Xsm randomized library or from the OYE

2.6 Y78 presequenced library. PCR was done using the same conditions mentioned for

the OYE 2.6 Y78Xsm-I113Xsm randomized library, as was the PCR workup and

transformation into ElectroTen-Blue® electrocompetent cells. Cells were plated onto LB-

amp agar plates and grown at 37°C for 36 h. Transformant cells were sequenced to

verify that both desired mutations were present at the ICBR complex using Sanger

sequencing.

Successful plasmid samples were then transformed into expression strain E. coli

BL21 (DE3) Gold cells using the same conditions mentioned for the OYE 2.6 Y78Xsm-

I113Xsm randomized library. After mutations were verified by sequencing, transformants

were assembled into a 96 well plate. Transformants were grown in 600 µL of LB-amp in

a 96 well plate overnight to reach saturation. The library was completed with the transfer

74

of 120 µL of saturated cultures into a new 96 well plate containing 30 µL of 80% glycerol

which brought the final concentration of glycerol to 15%.

Addressing concatomeric primer inserts

It was discovered that mutating the Y78 position was problematic. Using our

primer design and PCR method, it has been possible to produce mutations at position

Y78 on occasion, but far more often than not the PCR product contained concatomeric

primer inserts like the ones observed during the experiments with the OYE 2.6 Y78Xsm /

I113Xsm randomized library. It is also noteworthy to mention that randomized primers

(be they either KST or NNK) were far less likely to make concatomeric products than

the single mutant primers. Efforts were made to address the issue of concatomers at

position Y78. Neither altering the PCR program, varying the concentrations of the PCR

components, nor replacing any of the commercially purchased solutions was effective in

reducing the number of concatomeric PCR products. Multiple different stock solutions of

OYE 2.6 Y78 primer were purchased and never reduced the amount of concatomeric

products. As to the I113 position, no transformant ever sequenced contained multiple

primer inserts at that position. Therefore to work around this problem, OYE 2.6 Y78

single mutants already made were used as the templates for making the 36 double

mutants and as such, all PCR reactions used OYE 2.6 Y78 mutants as templates and

primers containing mutations at position I113 to make all the double mutants.

Construction of mutants in the OYE 2.6 Y78Xsm / I113C / F247X randomized libraries

Templates used to make the mutants in the OYE 2.6 Y78Xsm / I113C / F247X

randomized libraries were pBS2 derivatives obtained from the OYE 2.6 Y78Xsm /

I113Xsm presequenced library. Primers containing a NNK mix of nucleic acids at the

75

OYE 2.6 F247 position were used to make the OYE 2.6 Y78Xsm / I113C / F247X triple

mutants were from IDT (Appendix A, Table A-2). PCR was done using the same

conditions mentioned for the OYE 2.6 Y78Xsm / I113Xsm randomized library, as was the

PCR workup, the transformation into ElectroTen-Blue® electrocompetent cells and

purification of pooled plasmid samples. Degeneracy could be gauged for samples using

a NNK primer mix with the equation in Figure 1-11. Pooled plasmid samples with

sufficient degeneracy were used to transform an expression strain E. coli BL21 (DE3)

Gold cells. Three transformants per possible codon would be required to obtain a good

representation for each possible codon in a pooled plasmid sample. Therefore, 94

transformants were taken from each transformation of PCR sample using NNK primer

mix to accommodate the 32 codons possible in a NNK mix. Two wells were left

available for the OYE 2.6 Y78Xsm / I113C double mutant anchor and OYE 2.6 wt., which

were included in wells G12 and H12 respectively to be used as controls. Transformants

were grown in 600 µL of LB-amp in a 96 well plate overnight to reach saturation. The

library was completed with the transfer of 120 µL of saturated cultures into a new 96

well plate containing 30 µL of 80% glycerol was added to a final concentration of 15%.

Substrates

A list of substrates and products is shown if Figure 2-2.

Methyl 2-(hydroxymethyl)acrylate (1)

Methyl 2-(hydroxymethyl)acrylate was prepared in our lab by Bradford Sullivan28

using the method developed by Turki et al.32 Methyl 2-(hydroxymethyl)acrylate was

detected during screening by GC-FID using a Beta Dex 225 column (0.25 mm x 30 m).

The temperature program used began with an initial temperature of 100°C for 12 min,

followed by an increase at 20°C/min to a temperature of 180°C at which the program

76

remained for 5 min (GC method is listed as AZW3.Met in Appendix D). Methyl 2-

(hydroxymethyl)acrylate eluted near 11.8 min. The reduced S product eluted near 10.7

min and the reduced R product near 11.3 min.





column (0.25 mm x 30 m). The temperature program began with an initial temperature

of 140°C for 10 min, followed by an increase at 20°C/min to a temperature of 180°C at

which the program remained for 5 min (GC method is listed as AZW2.Meth in Appendix

D). 2-(Hydroxymethyl)-cyclohex-2-enone eluted near 13.1 min. The reduced S product

eluted near 10.2 min and the reduced R product near 10.8 min.





column (0.25 mm x 30 m). The temperature program used began with an initial



in Appendix D). 2-(Hydroxymethyl)-cyclopent-2-enone eluted near 13.1 min. The

reduced S product eluted near 11.4 and the reduced R product near 10.2 min.

Screening Assay

E. coli BL21 (DE3) Gold cells harboring plasmids containing OYE mutants of

interest were grown in a 96 well plate containing 600 µL of LB media with 200 μg/mL

77

ampicillin (LB-amp). Cells were grown at 37°C with 250 rpm of agitation overnight. The

saturated cultures were then used to inoculate a larger 2 mL square bottom 96 well

plate. This larger square bottom plate contained 600 µL of an auto-induction medium.

The auto-induction medium contained a mix of ZY media, 50x5052, 20x NPS, and 200

µg/mL ampicillin.33 Cells were induced in an aeration case developed at the University

of Florida by the machine shop in the Department of Chemistry. Induction occurred at

37°C with 350 rpm of agitation overnight. The increased agitation is required for

induction to occur. Induced cells were then separated from the auto-induction medium

by centrifugation. Auto-induction medium was removed and induced pelleted cells were

then resuspended in 600 µL of a reaction mixture. The reaction mixtures contained 50

mM KPi buffer with 100 mM glucose and 15 mM of the substrate of interest at pH 7.0.

Reactions were run at room temperature with 250 rpm of agitation overnight. Reactions

were quenched by adding 500 µL of ethyl acetate. The organic phase was separated by

centrifugation and extracted for analysis by GC.

Conclusions

It was previously shown during the OYE 1 W116 site saturation project that

position W116 is a significant site to use to engineer this protein. Modification at this

position has allowed new products to be obtained from using OYE 1 as a catalyst. The

ISM project showed that combing mutations at position at either I113 or F247 with the

linchpin mutation of Y78W in OYE 2.6 would allow that enzyme to make new exciting

products. This project proved that positions I113 and Y78 have substantially more room

for manipulation than previously shown. Regardless of which pair of the 36 double

mutations screened, the enzyme was still able to convert substrate to product. Also, the

OYE 2.6 Y78Xsm-I113Xsm presequenced library discovered a new linchpin cysteine

78

mutation at position I113. The I113C double mutants made in this project have better

conversion and provide better ee than the best triple and double mutants obtained from

the ISM project. And by targeting the west side of the OYE 2.6 active site we gained the

same additive benefits for our double mutants as were gained from the ISM project.

Also, the results of the OYE 2.6 Y78Xsm / I113C / F247X screening did imply that the

triple mutants that contained an Y78A, Y78C, or Y78V worked the best. These libraries

contained the most functional variants of the six libraries screened. The OYE 2.6 Y78C /

I113C / F247H and F247W triple mutants were the best mutants discovered during this

project, and the best variants discovered to date for using this enzyme to obtain the

desired (R)-5 product. Should this project be continued, a complete library of the F247

position that anchors off OYE 2.6 Y78C / I113C would need to be made and screened

against substrate 2. This screening could verify which mutant at that position is the best.

79

Table 2-1. List of the presequenced alkene reductase libraries made by the Stewart group.

Library Plasmid Description

Alkene Reductase pDJB5, pDB13, pDJB8, pDJB17, pDJB9, pDJB11, pDJB15, pDJB24, pDJB22, pDJB26, pDJB21, pDJB23, pDJB25, pDJB27, pDJB19, pDJB29

OYE 1, OYE 2, OYE 3, oye, OYEA, OYEB, NemA, ppNema, ppOYE, SeOYE, OPR1, OPR2, OPR3, Leopr, Ltb4dh, YNL134c

pDJB5 contains a T7 promoter, Kanr, and a GST-tag

All other plasmids contain T7 promoter, Ampr, and a GST tag

OYE 1 W116 pDJB5-W116X Non-tagged, GAL1/GAL10 promoter, Kanr

OYE 1 Y196 pET3b-OYE Non-tagged, T7 promoter, Ampr

OYE 2.6 Y78 pBS2 GST-tagged, T7 promoter, Ampr

excludes Y78C, Y78F, & Y78N

OYE 2.6 I113 pBS2 GST-tagged, T7 promoter, Ampr

OYE 2.6 Y193 pBS2 GST-tagged, T7 promoter, Ampr

OYE 2.6 Y78W / I113 pBS2 GST-tagged, T7 promoter, Ampr

OYE 2.6 Y78Xsm / I113Xsm

pBS2 GST-tagged, T7 promoter, Ampr

OYE 2.6 S388 pFB1 Non-tagged, T7 promoter, Ampr

OYE 3 W116 pRP4 Non-tagged, T7 promoter, Ampr

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Table 2-2. OYE 2.6 best variants discovered during the OYE 2.6 ISM studies.

Substrate Best variant for normal binding product

Best variant for flipped binding product

1 % conv 99, % ee 99 (S) OYE 2.6 wt % conv 99, % ee 91 (R) OYE 2.6 Y78W / F247A

2 % conv 99, % ee 99 (S) OYE 2.6 wt % conv 43, % ee 37 (S) OYE 2.6 Y78W / F247A

3 % conv 99, % ee 95 (S) OYE 2.6 wt % conv 97, % ee 98 (R) OYE 2.6 Y78W / I113V / F247H

(S)-(+)-Carvone % conv 48, % de 93 OYE 1 wt % conv 90, % de 99 OYE W116V

(R)-(-)-Carvone % conv 98, % de 97 OYE 1 wt % conv 78, % de 55 OYE W116A

Table 2-3. Q scores for NNK randomized libraries.

Library Q score Estimated number of amino acids obtainable from a transformation using this plasmid mix

OYE 2.6 Y78A / I113C / F247X 0.46 14.88

OYE 2.6 Y78C / I113C / F247X 0.77 18.83

OYE 2.6 Y78G / I113C / F247X 0.75 18.52

OYE 2.6 Y78S / I113C / F247X 0.75 18.52

OYE 2.6 Y78T / I113C / F247X 0.63 16.89

OYE 2.6 Y78V / I113C / F247X 0.61 16.68

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Figure 2-1. Flipped binding mode. This figure shows a 6-membered ring substrate undergoing catalysis in a flipped binding mode in OYE 2.6 wt. The silhouette of the 6-membered ring shows a normal binding mode.23

82

Figure 2-2. List of Chapter 2 substrates.

83

Figure 2-3. (S)-(+)-carvone bound in a flipped binding mode to the active site of OYE 1 W116I. The isopropylene group of (S)-(+)-carvone (blue) extends into the space between the residues at positions W116I and Y82. In the active site of OYE 1 W116I (S)-(+)-carvone can make use of this space due to the smaller isoleucine residue (PDB ID 4GE8).24

Figure 2-4. Mechanism of OYE 2.6. This figure shows the mechanism for the reduction of a bound α-unsaturated carbonyl substrate (black) by a reduced FMN (gold) in the active site of OYE 2.6 (violet). OYE 2.6 uses H188 and H191 as hydrogen bonding partners to lock the carbonyl into position.

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Figure 2-5. Diagram of the residues in the OYE 2.6 active site. All positions except L115, and Q248 were targeted during the ISM experiments. Positions Y78, I113, and F247 were also targeted during this project.57

Figure 2-6. Malonate bound in the active site of OYE 2.6 Y78W-I113C (PDB ID 4M5P).34

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Figure 2-7. Substrate 1 (pink) and 2 (green) modeled into the active of OYE 2.6 Y78W and OYE 2.6 wt. Bound 6 membered rings have difficulty maintaining a flipped binding mode in OYE 2.6 because they extend into the area around the Y78 positions. (PDB ID 4DF2 and 4QAI).34

OYE 2.6 GACAGATCCACTTTCCCAGGTACTTTGCTTATCACTGAAGCTACTTTTGTCTCTCCTCAA

Concatmoers GACGGATCCACTTTCCCAGGTGCTTTGCTTATCACTGAAGCTACTTTTGTCTCTCCTCAA

*** ***************** **************************************

OYE 2.6 GCCTCTGGTTATGAAGGTGCTGCTCCAG--------------------------------

Concatmoers GCCTCTGGTGCTGAAGGTGCTGCTCCAGGTAAGCCTCTGGCGGAGAAGGTGCTGCTCCAA

********* *****************

OYE 2.6 ----------------------------GTATTTGGACTGACAAGCACGCTAAAGCATGG

Concatmoers GCCTCTGGTGGTGAAGGTGCTGCTCCAGGTATTTGGACTGACAAGCACGCTAAAGCATGG

********************************

OYE 2.6 AAGGTTATTACTGATAAAGTTCATGCCAACGGTTCTTTCGTTTCAACCCAGTTGATTTTT

Concatmoers AAGGTTATTACTGATAAAGTTCATGCCAACGGTTCTTTCGTTTCAACCCAGTTGGTTTTT

****************************************************** *****

Figure 2-8. Sequence alignment of OYE 2.6 with a sample containing primer inserts. Mutation sections are shown in blue, sections with portions of the primer inserted are show in green and yellow.

86

Figure 2-9. Best variants discovered during both the ISM project and this project using a small residue matrix for obtaining the (R)-5 product from substrate 2.The small residue matrix library revealed that anchoring off I113C provided the best double mutants on the east side of the active site. Mutating a third position (F247) on the west side of the active site improved conversion even further. The triple mutants discovered to date are the OYE 2.6 Y78C / I113C / F247H and F247W mutants.

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Figure 2-10. OYE 2.6 Y78Xsm / I113Xsm library screening results for substrate 2. OYE 2.6 wt. is not shown but gave 100% conversion with 100% ee (S). The only double mutant combinations that provided any (R)-5 product were ones that used I113C mutants. And all I113 mutants gave some (R)-5 product, with The OYE 2.6 Y78C / I113C providing marginally the most.

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Figure 2-11. OYE 2.6 Y78A / I113C / F247X screening results for substrate 2. H12 is OYE 2.6 wt. and G12 is OYE 2.6 Y78A / I113C, the anchor double mutant. Nearly a third of the positions in this library gave conversion, and over half of those gave comparable (R)-5 to the anchor double mutant. Unfortunately, the positions sequenced were revealed to be the anchored double mutant.

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Figure 2-12. OYE 2.6 Y78C / I113C / F247X screening results for substrate 2. H12 is OYE 2.6 wt. and G12 is OYE 2.6 Y78C / I113C, the anchor double mutant. This library provided two of the best triple mutants with positions H4 and F5. Sequencing revealed that position F5 was an OYE 2.6 Y78C / I113C / F247H mutant and H4 was an OYE 2.6 Y78C / I113C / F247H mutant. These are the best variants discovered in this study, and the best discovered to date.

90

Figure 2-13. OYE 2.6 Y78G / I113C / F247X screening results for substrate 2. H12 is OYE 2.6 wt. and G12 is OYE 2.6 Y78G / I113C, the anchor double mutants. This library did not provide enough positions with sufficient conversion to warrant sequencing. Though this is not surprising since the OYE 2.6 Y78G / I113sm mutants provided the lowest conversions during the OYE 2.6 Y78sm / I113sm presequenced screening.

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Figure 2-14. OYE 2.6 Y78S / I113C / F247X screening results for substrate 2. H12 is OYE 2.6 wt. and G12 is OYE 2.6 Y78S / I113C, the anchor double mutants. This library did not provide enough positions with sufficient conversion to warrant sequencing.

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Figure 2-15. OYE 2.6 Y78T / I113C / F247X screening results for substrate 2. H12 is OYE 2.6 wt. and G12 is OYE 2.6 Y78T / I113C, the anchor double mutant. This library did not provide enough positions with sufficient conversion to warrant sequencing.

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Figure 2-16. OYE 2.6 Y78V / I113C / F247X screening results for substrate 2. H12 is OYE 2.6 wt. and G12 is OYE 2.6 Y78V / I113C, the anchor double mutant. Nearly a fourth of the positions in this library gave conversion, and nearly half of those gave comparable (R)-5 to the anchor double mutant. Unfortunately, the positions sequenced were revealed to be the anchored double mutant.

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Figure 2-17. Calculations for obtaining a Qscore of a pooled plasmid mix from a KST primer mix using data from a theoretical sequencing electropherogram. This figure shows a theoretical electropherogram with theoretical peak heights. a) The fraction of each peak height at a position over all peak heights at that position is shown on the left side of each peak. Peaks correspond to: blue/thymine, orange/guanine, green/adenine, purple/cytosine, and the dashed black represents perfect degeneracy. b) The peak heights of each base is used to estimate the amount of codons containing that base at that position. c) The sum of those estimates are used to obtain a Q value (QK or QS) for that position. d) The sum of the weighted QK and QS values is used to calculate the Qscore for the pooled mix. The Qscore approaches perfect degeneracy (equal amounts of bases at each position) as it approaches 1.0.26

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CHAPTER 3 IMPROVING THE THERMOSTABILITY OF OYE 2.6 THROUGH PROTEIN

ENGINEERING

Background

Improving Thermostability through Mutagenesis

Protein engineering is not exclusively used to alter enzyme chemistry; it can also

be used to improve enzyme thermostability.60–67 Several recent papers have reviewed

the methods used to improve the protein’s stability.59,68–73 This is due to the commonly

held belief that thermophilic proteins (thermostable proteins) are more stable than their

mesophilic (non-thermophilic) analaogs.59,74–76 The properties of naturally thermostable

proteins have become models for improving the stability of mesophilic proteins. One

notable property of naturally thermophilic proteins is that they tend to have very rigid

structures. This suggests that increasing the rigidity of a mesophilic protein might

increase its thermostability. One common way to identify less rigid positions in a protein

is to find residues with elevated B-factors (or B-values) in the crystal structure.77–79

Since these values are often used for gauging this promiscuity of atomic motion in a

crystal structure, they are also used to gauge a position’s flexibility and stability.

Targeting such positions for mutagenesis with subsequent screening for variants with

greater thermotabilities has become a standard method in protein engineering.

Iterative saturation mutagenesis (ISM) can be an ideal engineering strategy for

progressing through different variants toward a more thermostable protein. For

example, Reetz and coworkers successfully employed an ISM approach to improve the

thermostability of Bacilius subtilis lipase A.80 The most impressive example involved five

mutations that increased the enzyme’s T50 by nearly 50°C (T50 is the temperature at

which the enzyme’s conversion drops to 50% and is commonly used as the standard for

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evaluating thermostability in enzymes). This result was obtained from a project that

included ten 1st round libraries randomized by a NNK codon set. Positions that were

targeted were those with high B-factors in the crystal structure of lipase A. As part of

these efforts, the Reetz group developed a program called B-fitter to identify positions

with the highest B-factors in a protein crystal structures.

A directed evolution project aimed at increasing an enzyme’s substrate or

product range generally targets positions within the enzyme’s active site; by contrast

improving an enzyme’s thermostability often involves residues spread throughout the

protein structure and often on its exterior. Positions of interest are those believed to

impair the protein’s stability by their high local motion that could lead to global unfolding.

Residues with high B-factors in crystal structures are a logical starting point. In addition,

surface residues involved in subunit-subunit contacts within quaternary structure may

also play a large role in dictating protein thermostability.

In a study begun by Filip Boratynski in our group, we applied this approach to the

alkene reductase OYE 2.6 from Pichia stipitis. Three crystal structures of this enzyme

had been solved by a former group member (Yuri Pompeu), which allowed us to identify

promising residues by B-factor analysis. These efforts had two major goals. First, we

hoped to increase the thermostability of OYE 2.6 to make it more practically useful for

organic synthesis. Protein thermostability often correlates with organic solvent stability,

and the hydrophobic substrates of this enzyme act as organic solvents at the high

concentrations desirable for preparative synthesis. Our second goal was to test the

hypothesis that mutations of residues with high B-factors actually yielded proteins with

lessened motion at these positions. In other words, if protein thermostability has been

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improved, does the variant actually show a lower B-factor at that position? To the best

or our knowledge, while this is implied by the methodology’s rationale, it has never been

addressed experimentally.

Boratynski’s B-factor analysis of OYE 2.6 revealed that the C-terminal region had

significantly higher B-factors than the rest of the protein (Figures 3-1 and 3-2). He

therefore targeted the ten positions with the highest B-factors identified by the B-fitter

program for random mutagenesis using a NNK codon set (Figure 3-3). Each of the ten

1st generation libraries were screened at elevated temperatures to identify variants with

greater thermostability. From this study, the best were S388P and S388A, which

increased T50 by 3°C and 1°C respectively. In addition, both E389G and E389S

increased the T50 by 1°C. Boratynski created and screened a 2nd generation library

anchored by S388P and randomized at the adjacent E389. Unfortunately, no double

mutation with greater thermostability was discovered.

Project Summary

The somewhat disappointing results after preparing and screening ten 1st and

one 2nd generation libraries prompted us to pursue a more selective approach to

improving the thermostability of OYE 2.6. Boratynski had targeted the ten positions with

the highest B-factors and most of them were located on the C-terminal loop and thus in

the same area. We instead chose positions with unusually high B-factors compared with

their neighboring residues. We hypothesized that such positions showed high local

motion in an otherwise quiet region. This approach yielded four positions, which were

targeted for randomized mutagenesis (Figures 3-4 and 3-5). We also investigated

residues that appeared to be involved with the homodimerization of OYE 2.6. Five

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positions at the dimer interface of OYE 2.6 were randomized using a NNK codon set

(Figure 3-6).

Once created, all mutants were screened at elevated temperatures to identify the

most thermostable variants. The best from each library were subsequently purified for

more extensive characterization, with particular emphasis on determining their T50

values. Our hope was to identify one or more variants that could be combined with

others found earlier to create an OYE 2.6 mutant with significantly enhanced

thermostability.

Testing whether thermostable variants actually showed smaller B-factors

required solving X-ray structures. Our crystallization conditions were capricious and

many crystals had to be screened in order to find the few that diffracted well. We

therefore carried out a more extensive screening of crystallization conditions to identify

a more reproducible methodology. While these efforts failed to yield better crystallization

conditions, we were able to characterize one of our most thermostable OYE 2.6 variants

by X-ray crystallography.


Residues with High Local B-Factors

Three crystal structures of OYE 2.6 were available at the start of this project

(PDB ID: 3TJL, 3UPW, and 4DF2).34 We examined the B-factors of positions in these

structures and identified the twenty positions with the highest values. Ten had already

been targeted by Boratynski during this earlier study. These included the two terminal

positions (S2 and E405), two adjacent positions on the exterior of the protein (E298 and

E299), and six positions on the loop leading toward the C-terminus (M386, D387,

S388P, E389, E390, and V391). Examining the ten next highest B-factor values, we

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selected four residues that were flanked by sites with smaller B-factors. It was believed

that these positions may be causing local instability and if altered, may reduce global

instability throughout the enzyme. The positions selected were E41, D141, E145, and

K330. PCR was used with a NNK codon set to make individual saturation mutagenesis

libraries at each of the four positions. After successful PCR amplification and

subsequent transformation, plasmids from a pooled sample of transformants were

sequenced to estimate the degeneracy of the libraries at the targeted positions. Q

values were determined and the libraries were evaluated using the method developed

by Sullivan and Walton. Table 3-2 shows the Q values for all libraries made in this

project.26

Once libraries with adequate degeneracy had been obtained, plasmids were

used to transform the E. coli overexpression host and 95 randomly selected individual

clones were tested for the ability to reduce 2-methyl-cyclopenten-1-one, a good

substrate for the enzyme (substrate 1 in this chapter). Lysates of each library member

were heated for 15 min in a 47°C water bath prior to alkene reduction at room

temperature. This initial screening revealed twelve mutations that warranted more

careful investigation (Figure 3-7 through 3-16). These included E41S and E41P; D141E,

D141H, D141R and D141; E145A, E145G, E145M and E145T; K330D and K330T.

During the next stage of screening, protein concentrations were normalized and lysate

samples were subjected to a heat treatment at 42°C for 15 min (Figure 3-17). Eight of

the original twelve mutants gave results that suggested further study (E41S and E41P;

D141E, D141H and D141R; E145A, E145G and E145T). Lysates from these mutants

were tested for catalytic activity at room temperature after heat treatment along a

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temperature gradient to determine their T50 values (Figure 3-18 through 3-22). The only

variant that performed consistently better than wild type was the D141E mutant.

Dimer Interface Residues

After examining the OYE 2.6 crystal structure, we selected five positions that

appeared to be important to the protein’s quaternary structure. These positions all

contained hydrophobic residues and were located on the exterior of the protein at the

dimer interface between the two subunits of OYE 2.6 (I214, W244, L260, I311 and

F307). None of these five sites had significantly higher B-factor values than average.

Randomized libraries were made for each of these positions using a NNK codon library

as described above. After obtaining libraries with adequate Q scores from sequencing a

pooled plasmid sample, plasmids were used to transform the E. coli overexpression

host and 95 randomly-chosen clones were screened as before (Table 3-2). The only

mutant discovered from these efforts that warranted further investigation was the I311L

variant. Unfortunately, this mutant was not consistently better than wild-type OYE 2.6

and it was thus not pursued further (Figure 3-17). In summary, no mutant with greater

thermostability was discovered by targeting the dimer interface for mutagenesis.

Combining Thermostabilizing Mutations

After examining 19 individual positions in OYE 2.6 using saturation mutagenesis,

the next step was to combine the most beneficial changes with the goal of additivity or

ideally, synergism. Boratynski observed that changes to S388 yielded the greatest

increase in thermostability. Pro and Ala proved the best replacements. One problem

with this study was that three variants were not included in the original library from the

original PCR amplification. To ensure that all replacements for S388 were examined, we

used site-directed mutagenesis to add these “missing” variants to the collection (S388I,

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S388N and S388Y). Progressive screening (small scale, large scale, and heat treatment

gradient temperature assays) confirmed that the S388A and S388P mutants were

optimal for thermostability at that position (Figure 3-18).

Based on all previous work, the two best mutations for enhancing the

thermostability of OYE 2.6 were D141E and S388P. We therefore prepared the double

mutant and tested the protein using the heat treatment gradient temperature assay. The

double mutant was additively more thermostable when compared to the two parent

single mutants, yielding a T50 value of 44ºC (Figure 3-22). This is 3ºC higher than the

wild-type and represents the most thermostable OYE 2.6 variant that has been

reported.

Crystallization of OYE 2.6 D141E-S388P

As noted previously, the original crystallization conditions for OYE 2.6 have not

been as reproducible as desired. This requires setting up multiple redundant crystal

trials of the same protein under the same conditions to obtain diffraction-quality crystals.

It also requires screening multiple crystals from the same well to identify one that yields

high-resolution data. For this reason, Boratynski was unable to obtain crystallographic

data for the S388P single mutant of OYE 2.6.

In his initial studies, Yuri Pompeu found three sets of promising conditions in the

Qiagen Classics I and II kits. We systematically varied pH, protein concentration,

precipitant concentration and addition concentration around these three initial hits.

Optimizing around the conditions in well 71 (Qiagen Classics I Suite) involved a matrix

of precipitant concentrations (30%, 20%, 10%, 5% (w/v) PEG 8,000 MME), pH values

(6.0, 6.5, 7.0), and protein concentration (5, 10, 20, 40 mg/mL) in 0.1 M sodium

cacodylate, 0.2 M sodium acetate supplemented with 3% isopropanol. A separate study

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to optimize around conditions in well 91 (Qiagen Classics I Suite) used a matrix of

precipitant concentrations (30%, 20%, 10%, 5%, (w/v) PEG 5,000 MME), pH values

(6.0, 6.5, 7.0) and protein concentrations (5, 10, 20, 40 mg/mL) in 0.1 M Mes hydrate,

0.2 M (NH4)2SO4 with 3% isopropanol. Lastly, optimization around the original

conditions of well 27 (Qiagen Classics Suite II) utilized a matrix of precipitant

concentrations (3.5, 3.0, 2.4, 1.2, 0.6 M Malonate), pH values (6.0, 6.5, 7.0), protein

concentrations (5, 10, 20, 30, 40 mg/mL) and additive (4%, 3%, 2%, 1% isopropanol).

To our disappointment, none of these variations improved OYE 2.6

crystallization. Instead, in all cases these crystals would inevitably precipitate out of

solution as insoluble protein. Protein concentration was the only variable that had a

significant impact. Concentrations approaching 40 mg/mL provided fewer, larger

crystals with lower concentrations (ca. 10 mg/mL) afforded multiple, smaller crystals.

Though larger crystals obtained from the higher protein concentrations are of course

more desirable, they were often accompanied by a brown, insoluble protein precipitate.

Lower protein concentrations provided cleaner crystallization. One advance was that we

could use the hanging drop method to crystallize OYE 2.6; previously, only sitting drop

crystallization had been successful. This allowed us to obtain large numbers of crystals

for OYE 2.6 D141E / S388P.

We were able to obtain a high-resolution data set for the D141E / S388P mutant

to a resolution of 1.8 Å. Table 1 information for this structure can be found in Table 3-1.

After processing the data and solving the structure, we examined the C-terminal region

with particular interest. Proline substitution at position 388 did not appear to cause any

major structural perturbations to the C-terminal region. Our current working theory for

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the effect of the S388P mutation is that the more rigid proline may help lock the tail of

protein and thereby disfavor unfolding. Likewise, the D141E substitution did not impact

the structure significantly.

As noted previously, one key goal of this study was to determine when

successful mutations at positions selected for their high B-factors actually lower the B-

factors in the evolved protein. Our results were mixed. At position 388, the Pro residue

did indeed have a lower relative B-factor when compared to the wild-type Ser. On the

other hand, both the Asp and Glu residues at position 141 show essentially the same

relative B-factors, although the latter confers a 1ºC increase in T50.

The relative B-factor is the fraction of a position’s B-factor divided by the average

value of B-factors over the entire protein sequence. The relative B-factors of OYE 2.6

are highest near the C-terminus in all three of the previously-published structures

(Figure 3-23 through 3-28). In the structure of the D141E / S388P mutant, this peak in

the distribution of relative B-factors becomes a doublet with an indention at position 388.

In fact, using a normal distribution, the relative B-factor for position Ser 388 in OYE 2.6

wt. is 6 standard deviations greater than the mean of all relative B-factors (Figure 3-29).

By contrast, a Pro at position 388 has a relative B-factor to within 4 standard deviations

away from mean of all relative B-factors (Figure 3-30). While this remains a high local

value, it is clear that the mutation had a significant impact. For position 141 however,

this was not the case. The D141E mutation gave a slight improvement to OYE 2.6

thermostability, but did not significantly change the B-factor at that position. Using a

normal distribution, the relative B-factor for Asp at position 141 in wild-type OYE 2.6 is 2

standard deviations greater than the mean of all relative B-factors. A Glu at this position

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did not move the relative B-factor any closer towards the mean. It is tempting to

speculate that this lesser effect on B-factors is related to the mutation’s extremely

modest increase in T50 (1ºC).

Experimental

General



from Integrated DNA Technologies. All other reagents were obtained from commercial

suppliers and used as received. Plasmids were purified on small scales by Wizard®

minicolumns (Promega Life Sciences) and on large scales using CsCl density gradient

ultracentrifugation. DNA sequencing was carried out by the University of Florida ICBR

using capillary fluorescence methods using standard protocols. LB medium contained 5

g/L Bacto-Yeast Extract, 10 g/L Bacto-Tryptone and 10 g/L NaCl. ZY medium contained

5 g/L Bacto-Yeast Extract and 10 g/L Bacto-Tryptone. 50x5052 contained 25% glycerol,

2.5% glucose, and 10% a-lactose monohydrate. NPS x20 contained 66 g/L (NH4)2SO4,

136 g/L KH2PO4, and 142 g/L Na2HPO4.

Cloning

Construction of the plasmid used as a template for the OYE 2.6 thermal stability libraries

The plasmid used as a template to make all OYE 2.6 site saturation mutagenesis

libraries was pFB1 (Appendix E, Figure E-3), a pET22b derivative containing the gene

for wild-type OYE 2.6 with no extraneous affinity tags. It was made by the combined

efforts of Filip Boratynski and Bradford Sullivan. CsCl-purified pET22b and pBS2 were

digested with both NdeI and XhoI and the desired fragments were purified by low melt

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agarose gel electrophoresis. The two fragments were joined by T4 ligase and the

desired plasmid (pFB1) was identified after transformation into electro competent E. coli

cells.

Construction of OYE 2.6 libraries

Primers were designed to replace individual codons with a NNK mix (Appendix A,

Table A-3). This was carried out for E41, D141, E145, K330, I214, W244, L260, I311,

and F307. PCR was performed using 0.5 μL of 18 ng/μL pFB1 template, 5 μL of both 5

mM forward and reverse mutagenic primers, 1 μL of 10 mM dNTP mix, 10 μL of 5X HF

Phusion® Hot start buffer, 28 μL of sterile water, and 0.5 μL of 2 U/μL Phusion® Hot

Start II DNA Polymerase for a total reaction volume of 50 μL. PCR was performed using

a MJ Mini® thermocycler from BioRad. PCR amplifications were run with an initial

denaturation step at 98°C for 30 s, then a subsequent 25 cycles of denaturation at 98°C

for 10 s, annealing at 64°C for 30 s, and an extension step at 72°C for 3 min 30 s, after

which the reactions were completed with a final extension step at 72°C for 7 min 30 s.



with two 0.5 μL aliquots of 20 U/μL DpnI at 37°C to remove the parent template. DpnI

selectively targets hemi methylated DNA and as such is ideal for removing any

remaining template DNA from the PCR amplifications. The first aliquot of DpnI was

added immediately after PCR clean up and the second after 4 hours of digestion. After

DpnI digestion, samples were purified using Wizard® Plus SV Gel PCR Clean up kits by

Promega, using the manufacturer instructions.

The DNA samples were used to transform ElectroTen-Blue® electrocompetent

cells (ETB) using a BioRad Gene Pulser®. Electroporation was carried out with 4 μL of

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DNA sample and 50 μL of ETB cells at 2.5 kV. Electroporated samples were

immediately incubated in 600 μL of SOC medium at 37°C for 1 h. Cells were then plated

onto LB-amp agar medium (200 μg/mL ampicillin) and grown at 37°C for 36 h. The best

results were obtained from ETB daughter cells grown from the commercial stock on the

same day as the transformation would take place. Granddaughter cells provided fewer

transformants. Transformants were pooled and plasmid DNA extraction was performed

with Wizard® Plus minipreps DNA purification system by Promega, using the

manufacturer instructions. Pooled plasmid samples were then sequenced by ICBR

using Sanger sequencing. Electropherograms obtained from Sanger sequencing were

measured to estimate the samples degeneracy. Degeneracy could be gauged for

samples using a NNK primer mix with the equation in Figure 1-11 in Chapter 1. Pooled

plasmid samples with sufficient degeneracy were used to transform an expression strain

E. coli BL21 (DE3) Gold cells. To ensure complete coverage of the 32 possible codons

present in a NNK library, 95 randomly-chosen transformants were selected.

Transformants were grown in 600 μL of LB-amp in a 96 well plate overnight to reach

saturation along with the wild-type OYE 2.6 control. From these pre-cultures, 120 μL

was transferred into a new 96 well plate containing 30 μL of 80% glycerol. This brought

the final concentration of glycerol to 15% which is sufficient for cryoprotection of cells at

-80°C.

Substrates


2-Methyl-2-cyclopenten-1-one was purchased from Sigma Aldrich. 2-Methyl-2-

cyclopenten-1-one can be detected during screening by GC-FID using a DB-17 column

(0.25 mm x 30 m). The temperature program used began with an initial temperature of

107

60°C for 5 min, followed by an increase at 20°C/min to a temperature of 200°C at which

the program remained for 8 min. (GC method is listed as FB1.Meth in Appendix D). 2-

Methyl-2-cyclopenten-1-one eluted near 6.5 min. The reduced product eluted near 4.2

min. The chirality of the reduction product was not of interest to this project and thus

was not determined.

Screening

E. coli BL21 (DE3) Gold cells harboring pFB1 derivatives of interest were grown

in a 96 well plate containing 600 μL of LB-amp medium. Cells were grown at 37°C with

250 rpm of agitation overnight. The saturated cultures were then used to inoculate a

larger 2 mL square bottom 96 well plate. This larger square bottom plate contained 600

μL of ZYP-5052 auto-induction medium. This auto-induction medium contained a mix of

ZY medium, 50x5052 solution, 20x NPS solution, and 200 μg/mL ampicillin.81 Cells

were induced in an aeration case developed at the University of Florida by the machine

shop in the Department of Chemistry. Induction occurred at 37°C with 350 rpm of

agitation overnight. The increased agitation is required for induction to occur. Induced

cells were then separated from the auto-induction medium by centrifugation at 6°C for

10 min using 1,500 × g. Auto-induction medium was removed and cells were lysed

using freeze-thaw method. This involved freezing the plate at -80°C for 1 h followed by

thawing at room temperature for 30 min. After three rounds of freeze-thaw lysis, cells

were resuspended in 250 μL of lysis buffer containing 2 mg/mL of lysozyme and 100

mM KPi at pH 7.0. Lysis continued with an incubation in 37°C with 250 rpm agitation for

2 h. The crude lysates were heat-treated by incubating in a hot water bath at 47°C for

15 min. This temperature and time was selected because it was near the threshold for

inactivation of wild-type OYE 2.6 in this assay. An aliquot (200 μL) of heat treated cell

108

lysate was transferred to a new plate containing 250 µL of reaction mixture to determine

the remaining alkene reductase activity. The final assay concentrations were 5 mM

alkene substrate 1, 200 mM glucose, 44 U GDH, 0.3 mM NADP+, 100 mM KPi buffer at

pH 7.0. Reactions were run at 37°C with 250 rpm agitation for 3 h. Reactions were then

quenched by adding 500 μL of ethyl acetate. The organic phase was separated by


Large scale screening was performed on selected mutants of particular interest.

E. coli BL21 (DE3) Gold cells harboring pFB1 derivatives encoding OYE 2.6 mutants

were taken from streaks grown on a LB-amp agar plate. Cells were grown in 1 mL of

LB-amp for at least 6 h at 37°C with 250 rpm of agitation to reach saturation. After

saturation, 0.5 mL of inoculum was transferred to 30 mL of ZYP-5052 auto-induction

medium and induced overnight at 37°C with 350 rpm of agitation. After auto-induction

was complete, the cell cultures were pelleted by centrifugation, resuspended in 2 mL of

100 mM KPi buffer at pH 7.0, and lysed with two passes through a French Press. The

lysate was clarified by centrifugation and the pelleted debris was discarded. A Bradford

assay was used to determine the protein concentration of the supernatant. All mutant

protein samples were adjusted to 300 µg/mL by diluting with 100 mM KPi. An aliquot (50

μL) of the normalized protein solution was transferred to 250 μL of a reaction mixture

which had been preheated to 42°C using a thermocycler for 30 min. This temperature

was selected because it was at or slightly higher than the T50 of wild-type OYE 2.6. The

reaction mixtures contained 5 mM alkene substrate 1, 200 mM glucose, 44 U of GDH,

0.3 mM NADP+, and 100 mM KPi buffer at pH 7.0. The regeneration system used to

make NADPH which reduces the FMN of OYE and allows the protein to turnover is

109

shown in Figure 3-31. The heat treatment lasted 15 min after which the reactions were

removed from the thermocycler. Reactions were run at 37°C with 250 rpm agitation for

90 min, then quenched by adding 300 μL of ethyl acetate. The organic and aqueous

phases were separated by centrifugation and the organic phase was analyzed by GC-

FID.

OYE 2.6 mutants that converted as much or more substrate than wild-type OYE

2.6 were further evaluated using a temperature gradient assay. During this assay, cells

were grown, induced, lysed and normalized using the same method for the large scale

screening. Once the protein concentrations of all samples had been determined and

normalized to a common concentration, evaluation by heat treatment followed. The

reaction buffer mixture was preheated to the heat treatment temperature using a

thermocycler for 30 min. An aliquot (50 µL) of normalized protein solution was

transferred to 125 µL of a preheated reaction buffer mixture. Protein samples were heat

treated for 15 min before being removed from the thermocycler. 125 µL of reaction

mixture was added to each post heat treatment sample bringing the volume to 300 μL.

The now completed reaction mixtures contained 5 mM alkene substrate 1, 200 mM

glucose, 44 U of GDH, 0.3 mM NADP+, and 100 mM KPi buffer at pH 7.0. Reactions

were run at 37°C with 250 rpm agitation for 90 min. Reactions were quenched by

adding 300 μL of ethyl acetate, then the organic and aqueous phases were separated

by centrifugation and the organic phase was analyzed by GC-FID.

Protein Purification and Crystallization of OYE 2.6 Mutants

Protein purification for OYE 2.6 mutants was carried out using the procedure for

purifying the OYE 2.6 protein developed previously in our lab,34 which is a modification

of the procedure originally developed by Massey for purifying Saccharomyces

110

pastorianus OYE 1.6 E. coli BL21 (DE3) Gold cells harboring a derivative of pFB1

encoding the desired mutant were grown at 37°C in a 4 L New Brunswick Scientific M19

fermenter containing LB-amp. Cells were grown in the fermenter with 600 rpm of

agitation for 2 h to achieve mid log phase. Cells were induced by adding IPTG and

glucose to final concentrations of 0.4 mM and 100 mM, respectively. The culture was

grown at 30°C with 600 rpm of agitation for an additional 4 h. The culture was chilled at

4°C for 30 min before centrifugation at 5,000 g. Wet cell pellets were then resuspended

in 100 mM Tris-Cl buffer containing 10 μM PMSF at pH 8.0. Cells were then lysed under

12,000 psi with the aid of a French Press. Lysates were centrifuged at 15,000 g for 1 h.

Nucleotides were precipitated by adding protamine sulfate to a final concentration of 1

mg/mL and stirring at 4°C for 20 min. The supernatant was separated by centrifugation

at 15,000 g for 20 min, then protein was precipitated by adding 5 partitions of

ammonium sulfate every 5 min to achieve a final concentration of 78% saturation. The

protein precipitate was separated by centrifugation at 15,000 g for 1 h.

Purification of OYE 2.6 by an N-(4-hydroxybenzoyl) aminohexyl agarose affinity

column requires that the active site to be emptied of any bound ligand that would

interfere with binding to the phenol moiety of the column matrix. This was accomplished

by successive buffer exchanges during dialysis. The ammonium sulfate pellet obtained

from the salt cut was resuspended in 100 mM Tris-Cl, 100 mM (NH4)2SO4, 10 μM PMSF

buffer at pH 8.0. This was dialyzed against 1 L of 100 mM Tris-Cl, 100 mM (NH4)2SO4,

10 μM PMSF buffer at pH 8.0 overnight at 4ºC. The following day, the sample was then

dialyzed against 1 L of buffer containing 10 mM sodium dithionite for 2 h at 4ºC. After 2

h, the buffer was exchanged for a fresh 1 L of buffer containing 10 mM sodium dithionite

111

and dialysis continued for 2 h. The sample was then transferred to a fresh 1 L of buffer

without dithionite and dialyzed for 2 h, after which, the buffer was exchanged with a final

1 L of fresh buffer and dialyzed overnight. The final sample was then centrifuged at

15,000 g for 30 min to remove any insoluble debris accumulated during dialysis.

The affinity column was packed with 3 mL of the matrix and was equilibrated with

100 mM Tris-Cl, 100 mM (NH4)2SO4, 10 μM PMSF buffer at pH 8.0, prior to use. The

flow rate during purification was 0.5 mL/min. Elution of non-bound protein was

monitored by absorbance at 280 nm and a baseline established prior to loading protein

sample onto the column. Dialyzed protein samples were loaded onto the affinity column

in 10 mL portions. Binding of OYE 2.6 turned the column greenish brown. Bound OYE

2.6 was washed with starting buffer until 280 nm absorbance returned to the baseline;

this required anywhere between 30-60 mL of buffer. Protein was eluted by washing with

100 mM Tris-Cl, 100 mM (NH4)2SO4, 10 μM PMSF, 4 mM sodium dithionite at pH 8.0.

OYE 2.6 was then further purified by gel filtration with a Superdex 200 column

(Pharmacia) using 50 mM Tris-Cl, 50 mM NaCl buffer at pH 7.5. Pooled fractions

containing the desired protein were then concentrated by ultrafiltration using an Amicon

centrifugation tube to a final concentration of 40 mg/mL. Protein concentration was

determined by absorbance at 280 nm using an extinction coefficient (ε) and molecular

weights (MW) estimated by protparam (OYE 2.6 had an ε of 55,810 M-1cm-1 with a MW

of 45,274 Da).35

Crystals were grown using the published conditions.34 Wells contained 6 μL of

crystallization solution combined with 6 μL of 40 mg/mL protein in 50 mM Tris-Cl, 50

mM NaCl buffer pH 7.5 and used hanging drop vapor diffusion. The crystallization

112

solution contained 2.4 M malonate with 3% isopropanol at pH 7.0. The best crystals

obtained were grown in 12 days at 6°C. Crystals were soaked in a harvesting buffer of

2.4 M malonate at pH 7.0, 3% isopropanol with 15% (v/v) glycerol before being flash

cooled in liquid nitrogen and sent for data collection.

Data Collection and Structure Solution of OYE 2.6 D141E-S388P

The best crystals diffracted to a maximum usable resolution of 1.81 Å using the

X6A beamline at Brookhaven National Laboratory. The unit cell measured was 126.93

126.93 122.71 90 90 120 Å and the crystals belonged to space group P 63 2 2. The

asymmetric unit contained 1 molecule, a solvent content of 60.95%, and a Matthew’s

coefficient of 3.15 Å3/Dal.36

Reflection data were processed using the iMOSFLM program in the CCP4

program suite to a resolution of 1.81 Å.37 Phases were obtained using the Phaser-MR

utility of the PHENIX program suite by molecular replacement using a modification of P.

stipitis OYE 2.6 (PDB code 3TJL) as the search model.38 All ligands and water


showed one OYE 2.6 chain present in the asymmetric unit. The best solution for the

space group was determined to be P 63 2 2. The initial model was well defined

throughout the scaffold of the protein giving an Rfree value of 0.27. Further refinement

using the xyz coordinates, B-factors, real-space, and occupancies refinement strategy

features in PHENIX refine, as well as continued cycles of model building using the

structure validation tools in COOT, produced a model with an Rfree of 0.19. Once the

protein scaffold was established, the density in the OYE 2.6 active site was addressed.

Malonate from the crystallization buffer was present in the active site of most of the

OYE 2.6 structures and the D141E / S388P variant of OYE 2.6 was no exception.

113

Malonate fit the active site density well and was modeled into the active site as well as

additional areas throughout the model providing an Rfree of 0.18. The FMN cofactor was

modeled into the structure and subsequent refinement provided an Rfree of 0.17. This

was followed by further model work-up and refinement providing a final Rfree of 0.17.

B-Factor Data and Statistics

B-factors were evaluated as B-factor fractions. B-factors fractions were

calculated as the average of B-factors for all atoms of a position over the average of all

B-factors for every position. B-factors were determined to be significantly different by a

normal distribution test. Aspartate at position 141 in wild-type OYE 2.6 had a B-factor

fraction of 2.0, which was 3 standard deviations outside the average B-factor fraction of

all positions in the wild-type OYE 2.6 structure. A Glu at position 141 had a B-factor

fraction of 2.5, which remained 3 standard deviations outside the average B-factor

fraction of all positions in the D141E / S388P OYE 2.6 structure. This means that the

glutamate mutation did not significantly alter the B-factors at position 141. Position S388

had B-factor fraction of 2.9 in the wild-type OYE 2.6 structures, which was 6 standard

deviations outside the average B-factor fraction of all positions in the wild-type OYE 2.6

structure. The Pro at position 388 had a B-factor fraction of 3.9 which moved that

position to 4 standard deviations outside the average B-factor fraction of all positions in

the D141E / S388P OYE 2.6 structure. This means that the S388P mutation significantly

decreased the B-factor value at that position.

Conclusions

In this series of projects, we have discovered five mutations at three positions

that have consistently improved the thermostability of OYE 2.6 (D141E, S388A, S388P

E389G and E389S). Only the D141E / S338P OYE 2.6 double mutation was successful

114

at combining two positions for an additive effect. The combination of these two

mutations increased the T50 of OYE 2.6 between to 3°C, which was additive if not

synergistic. While these results are much more modest than those of Reetz, who

increased the thermostability of lipase A by 50°C, we have been able to both

successfully increase our protein’s stability and corroborate our hypothesis with

crystallographic data (Figure 3-29 and 3-30).80 B-factor data obtained from our crystal

structure proved that by improving the thermostability at position S388, we could

decrease the B-factor at that position relative to the protein. Whether there is a

correlation between the minor increases in thermostability general protein stability is yet

to be definitively tested. However, observations made during crystallization of this

protein suggest that this is the case. As mentioned earlier, while OYE 2.6 and some

OYE 2.6 variants can be crystallized using the published conditions; however, this was

not consistent, particularly for the mutants. This inability to crystallize interesting OYE

2.6 variants clearly limits us from structural investigations. On the other hand, mutants

from this study, selected for enhanced thermostability, crystallized very well and

consistently. This suggests that the D141E / S338P double mutant could be used as a

vehicle for examining crystal structures of other mutants developed by our lab that

proved refractory to crystallography.

Future attempts to increase the thermostability of OYE 2.6 should use a different

approach to mutagenesis. To date, our group has targeted over 33 positions in OYE 2.6

for single site saturation mutagenesis libraries (8% of the entire protein). Over half of

those positions were probed for the purpose of improving thermostability. Covering this

amount of ground would traditionally be done with using a technique like cassette

115

mutagenesis. Though it is less precise than single site mutagenesis, this type of

technique allows for a broader range of positions to be targeted. Given that we have

exhausted the rational and even semi-rational positions, this would be the ideal

approach of mutagenesis to use to further improve the thermostability of OYE 2.6.

116

Table 3-1. Crystallographic data collection and refinement statistics

OYE 2.6 D141E-S388P

X-Ray Source X6A beamline

Brookhaven National

Laboratory

Space Group P 63 2 2

Unit Cell Dimensions

126.93 126.93 122.71

90 90 120

a = b, c (Å)

Resolution (Å) 38.88 - 1.798

(1.862 - 1.798)

Unique Reflections 53506 (5254)

Completeness (%) 1.00 (1.00)

Multiplicity 11.8 (11.5)

Rsym 0.116 (0.632)

I/σ (I) 15.7 (4.0)

Rwork 0.1454 (0.1832)

Rfree 0.1697 (0.2048)

Ramachandran

Statistics

Favored (%) 97

Allowed (%) 3.2

Outliers (%) 0

Number of Protein,

Solvent, and Ligand

Atoms

3220, 481, 55

Average B Factors

(Å2) 20.93

Protein 19.64

Ligands 21.50

Solvent 29.50

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Table 3-2. Q scores for NNK randomized libraries.

Library Q score Estimated number of total amino acids obtainable from a transformation using this plasmid mix

OYE 2.6 E41X 0.78 18.95

OYE 2.6 D141X 0.71 18.02

OYE 2.6 E145X 0.86 20.00

OYE 2.6 K330X 0.56 15.99

OYE 2.6 I214X 0.65 17.19

OYE 2.6 W244X 0.68 17.57

OYE 2.6 L260X 0.65 17.19

OYE 2.6 F307X 0.73 18.33

OYE 2.6 I311X 0.76 18.67

118

Figure 3-1. The fraction of B-factors for each position over the average B-factors of all positions in the structure (B-factor fraction). The B-factors for each position are taken as an average of all atoms for that position obtained from the crystal structure. These atoms include both the residue atoms and the peptide backbone atoms for a position. Since the magnitudes of B-factors can differ across different structures, using a fraction that compares a position relative to the whole structure becomes more ideal. As the B-factor fraction of a position moves further and further above 1, it becomes easier to distinguish that position as one with a higher B-factor.

Figure 3-2. The relative B-factors of all three published OYE 2.6 wild type structures. The magnitude of B-factor are shown along a color gradient from blue with the lowest values, to yellow with higher values, to red with the highest values. Though the B-factor of a position may be different from structure to structure, the relative B-factor value for each position compared to the overall structure, is very consistent. If a position has a high B-factor value in one structure it will likely be high in all structures.

119

Figure 3-3. The positions targeted during the ISM thermostability project, their region in the protein, and their B-factors for structure 3TJL.

Figure 3-4. The positions targeted during the local maximum project, their region in the protein, and their B-factors for structure 3TJL.

120

Figure 3-5. The B-factor values for the positions targeted during the local maximum project. The B-factor values for positions target as well as those of the adjacent positions in three published structures of OYE 2.6 wt. These four positions were selected because they have higher B-factors than there adjacent positions and are considered local maxima of protein instability.

Figure 3-6. The positions selected for mutagenesis during the dimer interface project. OYE 2.6 is a homodimer and has a hydrophobic region connecting the two subunits. The top subunit is shown in purple while the second lower subunit is shown in orange. Leu 260 has two alternate conformations in the crystal structure.

3TJL 3UPWPDB Code

Position

L40

E41

22

38

B-factor

22 31

4DF2

D42

S140

A142

D141

K144

E145

A146

F329

T331

K330

42 53 55

27 28 36

33 34 41

34 35 40

48 53

38 44 51

25 25 35

32 30 37

25 26 36

21 23 33

32 39 51

121

Figure 3-7. The results for the small scale screening of the OYE 2.6 S388 library testing all 19 possible replacements with substrate 1.

Figure 3-8. The results of screening the OYE 2.6 E41X NNK randomized library.

122

Figure 3-9. The results of screening the OYE 2.6 D141X NNK randomized library.

Figure 3-10. The results of screening the OYE 2.6 E145X NNK randomized library.

1 2 3 4 5 6 7 8 9 10 11 12

A

B

C

D

E

F

G

H

5

OYE2.6 E145X NNK randomized library

Conversion 0% 1- 25% 25-50% 50-75% 75-99%

Color

Total 91 0 0 0

123

Figure 3-11. The results of screening the OYE 2.6 K330X NNK randomized library.

Figure 3-12. The results of screening the OYE 2.6 I214X NNK randomized library.

124

Figure 3-13. The results of screening the OYE 2.6 W244X NNK randomized library.

Figure 3-14. The results of screening the OYE 2.6 L260X NNK randomized library.

125

Figure 3-15. The results of screening the OYE 2.6 F307X NNK randomized library.

Figure 3-16. The results of screening the OYE 2.6 I311X NNK randomized library.

126

Figure 3-17. The results from the large scale screening assay. The libraries with mutations that warranted a large scale screening were: OYE 2.6 D141X, E145X, I311X, K330X, & S388P. Many successful reactions from the small scale screening (Hits) were revealed to be wild type (wt. Hits) after sequencing. The results of the sequencing are also shown in the bottom right corner.

127

Figure 3-18. The results of the best mutants from all ten ISM-libraries.

Figure 3-19. The results of the best mutants from OYE 2.6 E41X.

128

Figure 3-20. The results of the best mutants from OYE 2.6 D141X.

Figure 3-21. The results of the best mutants from OYE 2.6 E145X.

129

Figure 3-22. The results of the best mutants from all projects.

130

Figure 3-23. The positions targeted in both the ISM thermostability (circled in red) and local maximum projects (circled in yellow). Both projects targeted positions based on their B-factors. The region of Helix 25 is by far the area with the highest B-factor fractions. The most successful position targeted was OYE 2.6 S388P and is located near the pinnacle of that peak.

Figure 3-24. The relative B-factor fractions from the OYE 2.6 D141E-S388P structure. The conserved mutation at position D141 to a glutamate reduced the T50 by 1°C. This mutation was only marginally successful and did not impact the structure enough to alter the relative B-factor fraction at that position (circled in yellow). The mutation at position S388 to a proline reduced the T50 between 2-4°C and lowered the B-factor fraction of that position (circled in red).

131

Figure 3-25. The relative B-factor fraction of all OYE 2.6 positions along the 3UPW structure.

Figure 3-26. The relative B-factor fraction of all OYE 2.6 positions along the 3TJL structure. The pattern of B-factor fractions obtained from this structure is near identical to that of the 3UPW.

132

Figure 3-27. The B-factor fractions for all three published OYE 2.6 wt. structures.

Figure 3-28. The B-factor fractions of both the three OYE 2.6 wt. structures (blue, red, and yellow) and the OYE 2.6 D141E-S388P structure (purple). Though there is greater variance in the magnitude of the double mutant B-factor fraction the overall pattern is consistent. One significant difference is the broad “singlet” peak at helix 25 for the three wild type structures has becomes a “doublet” for the double mutant structure due the indent caused by S388P lowering the B-factor fraction.

133

Figure 3-29. The relative B-factor fractions of OYE 2.6 wt. in structure 3TJL. The standard deviation of the mean (σ) is 0.36. Position D141 has a B-factor fraction of 2.0 and is within 3 σ of the mean of all B-factor fractions. Position S388 has a B-factor fraction of 2.9 and is within 6 σ of the mean of all B-factor fractions.

Figure 3-30. The relative B-factor fractions of OYE 2.6 D141E-S388P structure. The standard deviation of the mean (σ) is 0.77. Position D141 has a B-factor fraction of 2.5 and is within 3 σ of the mean of all B-factor fractions. Position S388P has a B-factor fraction of 3.9 and is within 4 σ of the mean of all B-factor fractions.

134

Figure 3-31. The regeneration system used to make NADPH which reduces the FMN of OYE and allows the protein to turnover.

135

CHAPTER 4 THE STRUCTURE OF Saccharomyces cerevisiae OLD YELLOW ENZYME 3

Background

Crystallization of OYE Family Members

The crystal structure of Old Yellow Enzyme 1 from Saccharomyces pastorianus

(OYE 1) solved by Karplus and Fox in 1994 provided the first structural information on a

member of the OYE family.20 This structure identified key positions responsible for

binding FMN within the active site. These include T37, G72, Q114, and R243, which act

as either hydrogen bond donors or acceptors and L36, which makes hydrophobic

contacts with the C7 and C8 methyl groups of FMN (Figure 4-1). The crystal structure

also provided insight into the critical residues that interact with bound substrates. H191

and N194 form hydrogen bonds with the carbonyl of the bound substrate, which locks it

into position within the active site. These hydrogen bonds also stabilize the oxyanion

intermediate prior to its protonation (Figure 4-2). Other important residues were also

identified in OYE 1 active site (Figure 4-3). Our group has extensively studied site-

directed mutants of W116.22,23 Several residues on loop 6 (P295, F296, and L297)

might also play important roles since this loop lies on the southwest side of the active

site and is believed to be responsible for opening the active site to accommodate

NADPH binding / NADP+ release during the catalytic cycle (Figure 4-4).

In addition to our extensive work with S. pastorianus OYE 1, our group has also

explored several other OYE 1 homologs. Pichia stipites OYE 2.6 was used for a large

directed evolution study, and its crystal structure was solved by Yuri Pompeu as part of

these efforts.34 This information improved our mutagenic planning and also allowed us

to observe the effects of our mutations had on the OYE 2.6 structure. The structure of


136

OYE 2.6 showed very similar interactions between the protein and the FMN cofactor as

were observed previously for OYE 1 (Figure 4-1). In OYE 2.6, H188 and H191

appeared to be positioned to form hydrogen bonds with the substrate carbonyl (Figure

4-2). Other potentially important residues that might interact with the substrate

(analogous to those in OYE 1) were also revealed (Figures 4-3 and 4-4).

Old Yellow Enzyme 3 (OYE 3) is an OYE homolog from Saccharomyces

cerevisiae originally discovered by the Massey group during their efforts to clone the

gene encoding S. pastorianus OYE 1.9 OYE 3 shares significant sequence similarity to

OYE 1 (80%). We had included OYE 3 in our original collection of overexpressed OYE

1 homologs and this enzyme has also been explored by others (Figure 4-5).56 Our

collaborators (Professor Elisabetta Brenna) was interested in OYE 3 because it yielded

products with opposite stereoselectivites for some substrates when compared to OYE 1

and OYE 2.52 Because of the very high sequence identity between OYE 3 and OYE 1,

we decided to solve the crystal structure of the former to understand its divergent

stereoselectivity. In addition, we also explored the impact of replacing W116 in OYE 3

since the corresponding changes in OYE 1 greatly impacted stereoselectivity.

Project Summary

One goal of this project was to crystallize and solve the crystal structure of S.

cerevisiae OYE 3. We initially attempted to crystallize a GST-OYE 3 fusion protein. The

GST tag simplifies protein purification and allows for a similar high throughput isolation

strategy analogous to that of a His-tag.82 A GST-OYE 3 fusion protein would stream-line

the cloning and allow us to make constructs for both easy protein purification and

crystallization. In parallel, we also explored crystallization conditions for the native OYE

3 protein (lacking the GST-tag). We also explored mutagenesis of W116, the analogous



137

position to W116 in OYE 1 and I113 in OYE 2.6 (the significance of these two positions

was mentioned extensively in Chapter 2). We created a single 96 well plate containing

the complete collection of OYE 3 W116 mutants and then screened these variants

against a set of substrates (Figure 4-6 through 4-8). Finally, we also attempted to

crystallize two OYE 1 double mutants related to the sequence of OYE 3 (OYE 1 W116A

/ F296S and W116V / F296S). Because neither OYE 1 double mutant yielded usable

crystals, we carried out the analogous study using two OYE 3 single mutants (W116A

and W116V).


Crystallization of OYE 3

We first explored the possibility of crystallizing S. cerevisiae OYE 3 as a GST-

fusion protein since this would greatly simplify protein isolation. We successfully purified

the target fusion protein by our standard methods (glutathione affinity chromatography

followed by gel filtration). Crystallization trials were set up using several commercial

screening kits; however, few conditions provided even quasi crystals of sufficient quality

to warrant further optimization. Based on these results, the best initial hit conditions

involved 5 mg/mL GST-OYE 3 in 100 mM Citrate, 20% (v/v) PEG 4000, 18%

isopropanol, pH 5.5 and grown at room temperature for 18 days. Unfortunately even

these conditions provided only quasi crystals which were not close to diffraction quality.

After these unsuccessful results, we turned our attention to crystallizing native OYE 3.

We subcloned the OYE 3 gene from the GST-tagged construct in plasmid pDJB6

by a process that involved several site-directed mutations to remove undesired

restriction enzyme sites. The resulting gene was introduced into plasmid pET22b for

native overexpression (plasmid pRP4). We carried out a small scale preliminary study to

138

predict whether OYE 3 would bind to the phenol affinity column originally developed to

purify native OYE 1. We tested the ability of OYE 3 to reduce 5 mM 2-methyl-

cyclohexen-1-one (substrate 12) in the presence and absence of 5 mM 4-chlorophenol

(Figure 4-9). This phenol commonly inhibits OYE homologs; moreover this is the same

functional group found on our affinity column. Cells harboring pRP4 were grown until

they reached mid log phase then they were induced overnight using auto-induction

media.81 Cells were harvested and lysed by the addition of lysozyme. Lysates were

combined with KPi buffered reaction mixtures, which were run overnight. Reactions

were quenched with ethyl acetate and the organic phase was then extracted for

analysis by GC-MS. The reactions lacking 4-chlorophenol showed complete reduction

of the enone while those containing 4-chlorophenol showed no conversion. These

results implied that the phenol affinity column developed for OYE 1 would also be

successful for OYE 3.

Native OYE 3 was overexpressed in E. coli and purified by ammonium sulfate

fractionation followed by our standard affinity chromatography followed gel filtration

methods used for OYE 1. After screening approximately 300 crystallization conditions,

the only viable crystals were observed when low molecular weight PEG was the main

precipitant at near neutral pH values (pH 6). Further attempts to optimize around other

promising conditions were unsuccessful, producing either showers of aggregated

crystals or non-crystallized precipitant. The most promising initial conditions were

obtained using the PEGRx HT screening kit from Hampton research in wells B12, C6,

C9, and G3. All produced showers of microcrystals with spherical nucleation sites. Wells

B5, C5, C8, D5, and E6 all produced large crystals of better quality with fewer, but still

139

multiple, nucleation sites. Conditions from wells A8 (100 mM MES monohydrate, 22%

v/v PEG 400, pH 6.0), A10 (100 mM sodium citrate tribasic dehydrate, 30% v/v

polyethylene glycol monomethyl ether 550, pH 5.0), and B6 (100 mM HEPES, 30%

PEG 1,000, pH 7.5) produced the highest quality of crystals with only single nucleation

sites. Crystals obtained from these conditions were rhombus shaped (Figure 4-10) and

had the gold color which is characteristic of both OYE 1 and OYE 2.6 crystals. We

attempted to optimize around the conditions in well A10 by arraying a matrix of

precipitants (15% v/v PEG 8,000, 18% v/v PEG 3,500, & 25% v/v PEG 400), pH values

(4.5, 5.0, 5.5, & 6.0) and different protein concentrations (20 and 10mg/mL). We also

attempted to optimize around the conditions in well B6 by arraying a matrix of

precipitants (30% v/v PEG 1,000, 24% v/v PEG 1,500, & 12% v/v PEG 3,000), pH

values (6.5, 7.0, 7.5, & 8.0) and protein concentrations (20 and 10 mg/mL). None of

these efforts yielded better-quality crystals. In fact, the further the conditions deviated

from the initial screening conditions, the worse the crystals became. We therefore chose

crystals for data collection from the A8 and A10 wells.

The most successful conditions were 100 mM MES monohydrate, 22% v/v PEG

400, pH 6.0 which provided crystals that diffracted to a maximum usable resolution of

1.8 Å using the 21-ID-G beamline at the Advanced Photon Source, Argonne National

Laboratory. The unit cell measured 61.214 107.762 141.071 Å and the crystals

belonged to space group P 21 21 21. The asymmetric unit contained two molecules and

a solvent content of 53.52% with a Matthews coefficient of 2.65 Å3/Dal.36

Data Reduction and Structure Solution

Reflection data for the OYE 3 structure without ligand were processed using

XDS83 to a resolution of 1.8 Å. Phases were obtained using the Phaser-MR utility of the

140

PHENIX program suite38 by molecular replacement using a modification of S.

pastorianus OYE 1 (PDB code 1OYB) as the search model. All ligands and water


showed two OYE 3 chains present in the asymmetric unit. The best solution for the

space group was determined to be P 21 21 21 and the initial model was well defined

throughout the scaffold of the protein giving an Rfree value of 0.37. The C-terminal region

at both positions K398 and N399 was a notable exception and these two positions were

therefore removed from the initial search model. The initially calculated 2Fo-Fc and Fo-Fc

maps showed electron density patterns that could be easily identified as FMN. After one

round of simulated annealing refinement, the Rfree value dropped to 0.31. Further

refinement using the xyz coordinates, B-factors, real-space, and occupancies

refinement strategy features in PHENIX.refine as well as continued cycles of model

building using the structure validation tools in COOT produced a model with an Rfree of

0.22. Once the protein scaffold was established, the electron density in the OYE 3

active site was addressed. The FMN cofactor was modeled into the structure and

subsequent refinement provided an Rfree value of 0.19. Components from both the

crystallization conditions and the purification protocol were modelled into the active site

to identify any ligand present; however, none reasonably accounted for the electron

density observed by the 2Fo-Fc and Fo-Fc maps. Final refinement with chloride modeled

into the active site provided an Rfree value of 0.19.

Reflection data for OYE 3 with HPBA were processed using XDS83 to a

resolution of 1.9 Å. Phases were obtained using the Phaser-MR utility of the PHENIX

program suite38 by molecular replacement using a modification of S. pastorianus OYE 1

141

(PDB code 1OYB) as the search model. All ligands and water molecules were removed

prior to molecular replacement. The best solution for the space group was determined

to be P 21 21 21 with an initial Rfree value of 0.38. Terminal positions K398 and N399

were truncated from model and one round of refinement using simulated annealing

followed reducing the Rfree value to 0.33. Further refinement using the xyz coordinates,

B-factors, real-space, and occupancies refinement strategy features in PHENIX.refine

as well as continued cycles of model building using the structure validation tools in

COOT reduced the Rfree to 0.25. The FMN cofactor was modeled into the structure and

subsequent refinement dropped the Rfree value to 0.24. Once the protein scaffold was

established, the electron density in the HPBA was modeled into the active site provided

and subsequent refinement reduced the Rfree value to 0.23. Further modeling lowered

the Rfree value to 0.22.

Reflection data for OYE 3 W116V were processed using XDS83 to a resolution of

1.9 Å. Phases were obtained using the Phaser-MR utility of the PHENIX program suite38

by molecular replacement using a modification of S. pastorianus OYE 1 (PDB code

1OYB) as the search model. All ligands and water molecules were removed prior to

molecular replacement. The best solution for the space group was determined to be P

21 21 21 with an initial Rfree value of 0.39. Terminal positions K398 and N399 were

truncated from model and one round of refinement using simulated annealing followed

reducing the Rfree value to 0.33. The FMN cofactor was modeled into the structure and

subsequent refinement reduced the Rfree value to 0.32. Further refinement using the xyz

coordinates, B-factors, real-space, and occupancies refinement strategy features in

PHENIX.refine as well as continued cycles of model building using the structure

142

validation tools in COOT dropped the Rfree to 0.24. Once the protein scaffold was

established, the electron density in the OYE 3 active site was addressed. No HPBA

ligand was bound within the active site, instead the active site had water analogous to

that of the OYE 1 structure 1OYA and 1OYC. Final refinement with water modeled into

the active site provided an Rfree value of 0.24.

The overall structure of OYE 3 is nearly identical to that of OYE 1. OYE 3

structure has an analogous alpha beta barrel with the active site located within the

barrel (Figure 4-11). The FMN environment of OYE 3 was similar to that of OYE 1, with

amino acids T37, G72, Q114, and R243 hydrogen bonding to the FMN (Figure 4-1).

Residues H191 and N194 were positioned in a way that would allow them to form

hydrogen bonds to the carbonyl of a bound substrate, analogous to their roles in OYE 1

(Figure 4-2). Interestingly, nearly all active site residues were identical between OYE 1

and OYE 3, with the exception of position 296 (Ser in OYE 3 and Phe in OYE 1). A

complete list of the measured distances between β-carbons of each active site residue

to the closed carbon on the bound ligand are listed in Table 4-3. Position 296 is located

on loop 6, which opens during the catalytic cycle. Phenylalanine 296 in OYE 1 extends

into the active site when the loop is closed, taking up significantly more space than

S296 in OYE 3, which directs its side-chain outward, toward the external solvent (Figure

4-12). Efforts to understand how this sequence difference at position 296 impacts the

stereoselectivities of OYE 1 and OYE 3 are ongoing and involve molecular dynamics

simulations of both enzymes (a collaboration with Professor Adrian Roitberg and his

group).

143

OYE 3 W116 Site Saturation Mutagenesis

After successfully solving the crystal structure of OYE 3, we created all possible

variants at position 116 using the non-GST-tagged OYE 3. The wild-type and all

mutants were screened against a series of substrates that had been investigated in

previous studies in order to obtain comparative data (Figure 4-15 through 4-28).22,24,58

In the case of cyclopentenone 1, OYE 3 W116H and W116Y showed the best

conversion and (S)-selectivitivty while OYE 3 W116E provided almost racemic product

(the greatest level of (R)-product observed from the mutant collection). Wild-type OYE 3

showed no significant conversion for 1.

In the case of cyclohexenone 2, OYE 3 W116H and W116Q yielded the most

(S)-product while OYE W116E afforded the highest level of (R)-product. As before wild-

type OYE 3 did not reduce cyclohexenone 2.

Neither wild-type nor any W116 mutant reduced pulegone 3. In the case of (S)-

and (R)-carvone (4 and 5),the wild-type and all W116 variants gave the same

stereochemical outcomes. This stands in marked contrast to the results with OYE 1, for

which changing W116 dramatically altered the stereochemical course of carvone

reductions.24 This points to an interplay between the residues at position 116 and 296,

despite the long distance between them (18.6 Å).

Neither wild-type OYE 3 nor any of the W116 mutants gave significant

conversion for substrates 11, 13, 14 or 15. Enones 21 and 22 were efficiently reduced

by OYE 3 wild-type and all W116 variants; however, highly hindered substrates 23 and

24 gave poor conversion with the same proteins. The best were OYE 3 W116P and

W116T for cyclohexenone 23 and OYE 3 W116C for spirocycle 24. While many of the

144

OYE 3 W116 mutants gave conversion for substrate 25 (with the best provided by the

W116N variant), none yielded the opposite stereoisomer.

X-Ray Crystallography Studies of OYE 3 W116 Mutants and Related Proteins

Our collaborators asked us to solve the structures of two OYE 1 variants whose

sequences were related to that of OYE 3: OYE 1 W116A / F296 S and W116V / F296S.

Unfortunately, neither of these double mutants bound to the phenol affinity column

sufficiently strongly to allow purification. Instead, both eluted from the column prior to

dithionite addition. This behavior suggests significant changes to the active site

structure and / or the local environment of the FMN. The small fraction of protein that

did bind to the affinity column was collected and further purified by gel filtration. The

isolated proteins immediately precipitated from solution after mixing with the standard

OYE 1 crystallization buffer, yielding a brown, insoluble mass. His-tagged OYE 1

mutants were subsequently tested, but fared no better under our standard OYE 1

crystallization conditions. We also used an older purification protocol for OYE 1 based

on anion exchange chromatography.5 Unfortunately, the double mutants were not

sufficiently robust to survive this purification and a significant amount of the protein

precipitated prior to transfer to crystallization buffer. The remainder precipitated in the

crystallization buffer itself.

Interestingly, the single mutant OYE 1 F296S bound avidly to the phenol affinity

column and crystallized without incident. Moreover, our group has previously solved the

crystal structures of the single mutants OYE 1 W116A and OYE 1 W116V without

incident.24,84 We must therefore conclude that the combined effect of mutations at

positions 116 and 296 affect the active site so significantly in OYE 1 that they prevent

phenol binding and significantly diminish protein stability.

145

Because OYE 3 can be considered (at least formally) analogous to the OYE 1

F296S single mutant, and we had previously prepared clones for OYE 3 W116A and

W116V, we used these variants as models for the OYE 1 double mutants. Both OYE 3

mutants could be purified by phenol affinity chromatography and crystallized under the

standard conditions developed for wild-type OYE 3. Crystals of OYE 3 W116V were

soaked with p-hydroxybenzaldehyde and sent for data collection. The OYE 3 W116V

crystal successfully diffracted and the structure was determined. Unfortunately the

soaking was unsuccessful and p-hydroxybenzaldehyde did not bind to the active site.

For comparison, we overlapped the OYE 3 structure with p-hydroxybenzaldehyde

bound to the OYE 3 W116V structure. Substitution of valine at position W116 decreased

the distance from the C2 of p-hydroxybenzaldehyde to the β-carbon of the residues by

0.1 Å (Figure 4-13). With a valine mutation however, the distance from the edge of the

residue (γ-carbon for valine and the ζ-carbon for tryptophan) to the C2 of p-

hydroxybenzaldehyde increased by 2.7 Å.

OYE 1 F296S was also crystalized and solved for comparison. Crystals of OYE 1

F296S were also soaked with p-hydroxybenzaldehyde and sent for data collection. The

crystal successfully diffracted and the structure was determined. Unfortunately the

soaking was unsuccessful for OYE 1 F296S as well and p-hydroxybenzaldehyde did not

bind to the active site. The serine at position F296S is orientated in the same manner as

position S296 in OYE 3. There appears to be no significant difference in the orientation

of loop 6 in OYE 1 F296S due to the substitution of a serine. For comparison, we

overlapped the OYE 1 structure with p-hydroxybenzaldehyde bound (1OYB) to the OYE

1 F296S structure and OYE 3. Substitution of serine at position F296 made no

146

difference in the distance (7.8 Å) from the C4 of p-hydroxybenzaldehyde to the β-carbon

of the 296 residues (Figure 4-14). With a serine mutation however, the distance from

the edge of the residue (β-carbon for serine and the ζ-carbon for phenylalanine) to the

C4 of p-hydroxybenzaldehyde increased by 3.2 Å.

Experimental

General



from Integrated DNA Technologies. Crystallography screening kits (Classics Suite/

AmSO4 and PEGRx HT) were purchased from Qiagen and Hampton Research,

respectively. All other reagents were obtained from commercial suppliers and used as

received. Plasmids were purified on small scales by Wizard® minicolumns (Promega

Life Sciences) and on large scales using CsCl density gradient ultracentrifugation. DNA

sequencing was carried out by the University of Florida ICBR using capillary

fluorescence methods using standard protocols. LB medium contained 5 g/L Bacto-

Yeast Extract, 10 g/L Bacto-Tryptone and 10 g/L NaCl. ZY medium contained 5 g/L

Bacto-Yeast Extract and 10 g/L Bacto-Tryptone. 50x5052 contained 25% glycerol, 2.5%

glucose, and 10% a-lactose monohydrate. NPS x20 contained 66 g/L (NH4)2SO4, 136

g/L KH2PO4, and 142 g/L Na2HPO4.

Cloning

Construction of plasmid used for libraries

Plasmid construction began with pDJB6, a construct made by the combined

efforts of former group members Iwona Kaluzna and Despina Bougioukou. This plasmid

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was constructed from pIK2 (a pYEX-41 derivative containing a GST tag) with pET26a

and adding the wild-type S. cerevisiae OYE 3 gene.85 In this study, a silent mutation

was made at position Y389 in the OYE 3 gene to remove an internal NdeI site (primers

are listed as Y389Y Fwd and Rev in Appendix A-4).26,27 The PCR product was purified

by a Wizard® Plus SV Gel PCR Clean up kit (Promega), transformed into ElectroTen-

Blue® E. coli cells and the desired silent mutation was verified by Sanger sequencing.

The resulting plasmid was designated pRP1. Cloning continued with the removal of a

second internal NdeI site which preceded the GST gene using PCR. This yielded

plasmid pRP2. A unique NdeI site was introduced at the start of the OYE 3 coding

region using PCR, resulting in plasmid pRP3. Plasmid pRP3 was prepared on a large

scale by CsCl density gradient29 and the gene encoding the native OYE 3 protein was

excised by digesting with NdeI and XhoI. The fragment was inserted into pET22b,

previously cut with NdeI and XhoI, using T4 DNA ligase to yield pRP4 (Appendix E,

Figure E-4). Products from the ligation reaction were transformed into ETB cells and

sequenced to verify the desired sequence of pRP4. This was used to transform E. coli

overexpression strain BL21 (DE3) Gold using electro-transformation.

Construction of an OYE 3 W116 site-saturation mutagenesis library

Plasmid pRP4 was the template used to make W116 mutants in OYE 3. A set of

mutagenic primers containing a single codon replacement at position W116 was used to

make each of the 19 mutants (primers are listed in Appendix A, Table A-4). PCR was

performed using 0.5 μL of template (18 ng/µL), 5 μL of both forward and reverse

mutagenic primers (5 mM), 1 μL of dNTP mix (10 mM), 10 μL of 5X HF Phusion® Hot

start buffer, 28 μL of sterile water, and 0.5 μL of Phusion® Hot Start II DNA Polymerase

(2 U/μL) for a total reaction volume of 50 μL. PCR was performed using a MJ Mini®

148

thermocycler from BioRad and samples were run with an initial denaturation step at

98°C for 30 s, followed by 25 cycles of denaturation at 98°C for 10 s, annealing at 64°C

for 30 s, and an extension step at 72°C for 3 min 30 s, after which the reactions were

completed with a final extension step at 72°C for 7 min 30 s.

All PCR samples were purified by Wizard® Plus SV Gel PCR Clean up kits

(Promega), using the manufacturer instructions. Samples were then incubated overnight

with two 0.5 μL aliquots of 20 U/μL DpnI at 37°C to remove the parent template. The

first aliquot was added immediately after PCR clean up and the second was added after

4 h of digestion. After DpnI digestion, samples were purified using Wizard® Plus SV Gel

PCR Clean up kits.

DNA samples were used to transform E. coli ETB cells using a Gene Pulser®

from BioRad. Electroporation was carried out with 4 μL of DNA sample and 50 µL of

ETB cells with a voltage of 2.5 kV. Cells were then incubated in 600 μL of SOC medium

at 37°C for 1 h before plating onto LB-amp agar plates and growing at 37°C for 36 h.

Plasmids from randomly-chosen colonies were sequenced to verify that the desired

mutation was present using Sanger sequencing. The desired plasmids were then used

to transform E. coli expression strain BL 21 (DE3) Gold. This was accomplished using

electroporation (2.5 kV) with 4 μL of plasmid (10 ng/μL) and 80 μL of E. coli BL21 (DE3)

Gold electrocompetent cells. Electroporated samples were incubated in 600 μL of SOC

medium at 37°C for 45 min prior to plating onto LB-amp agar plates and growing

overnight at 37°C. Mutations were verified by sequencing, then transformants were

assembled into a 96 well microtiter plate. Transformants were grown in 600 μL of LB-

amp in a 96 well microtiter plate overnight to reach saturation. The library was

149

completed with the transfer of 120 μL of saturated cultures into a new 96 well microtiter

plate containing 30 μL of 80% glycerol which brought the final concentration of glycerol

to 15%.

Testing of Phenol Binding to OYE 3

E. coli harboring pRP4 were taken from cell streaks on an LB-amp agar plate and

grown in 5 mL of LB-amp overnight to reach saturation. A 500 μL aliquot of this

preculture was used to inoculate a solution containing 50 mL of ZYP-5052 auto-

induction media and grown overnight. Cells were harvested and resuspended in 5 mL of

50 mM KPi, pH7 containing 1 mg/mL lysozyme. Cells were incubated in the lysozyme

solution for 1 h at 37°C. Samples were then centrifuged at 12,000 × g to remove the

insoluble debris. An aliquot of 200 μL of lysate was added to a pair of 500 μL reaction

mixtures containing 50 mM KPi, pH 7, 5 mM substrate 12, 100 mM glucose, 44 U GDH,

0.3 mM NADP+. One of the two reaction mixtures was supplemented with 5 mM 4-

chlorophenol. Reactions were run overnight at 30°C before quenching with 0.5 mL of

ethyl acetate. The organic phase was extracted and analyzed by GC-MS by method

JON.Meth (Appendix D) using a DB-17 column.

GST-OYE 3 Fusion Protein Purification and Crystallogenesis

E. coli BL21 (DE3) harboring plasmid pDJB6 (a derivative of pET22b with an

GST-OYE 3 fusion coding region flanked by NdeI and XhoI sites) was grown at 37°C in

a 4 L fermenter containing LB medium supplemented with 36 µg/mL kanamycin (LB-

kan). Cells were grown in the fermenter with 600 rpm of agitation for 2 h to achieve mid

log phase then induced by adding IPTG to a final concentration of 0.4 mM and grown

with 100 mM glucose at 30°C with 600 rpm of agitation for 3 h. The cell solution was

then chilled at 4°C for 30 min before centrifugation at 5,000 × g. The wet cell pellet was

150

then resuspended in 1 mL of 1× PBS buffer per gram of wet cell pellet. Cells were then

lysed under 12,000 psi with the aid of a French Press. The lysate was centrifuged for 1

h at 15,000 × g. Portions of 10 mL of the supernatant were loaded onto a glutathione

column charged with 1× PBS buffer. Samples were eluted with 20 mL of an elution

buffer containing 50 mM Tris with 3 mg/mL of reduced glutathione at pH 7.5. The eluant

was further purified by gel filtration with a Superdex 200 column (Pharmacia) using 50

mM Tris-Cl, 50 mM NaCl, pH 7.5. Pooled elutant samples were then concentrated by

ultrafiltration to a final concentration of 20 mg/mL using Amicon tubes. Protein

concentration was determined by absorbance at 280 nm using an extinction coefficient

(ε) and molecular weights (MW) estimated by protparam (GST-OYE 3 fusion protein

had an ε of 106,480 M-1cm-1 with a MW of 68,543 Da).35

Protein crystals were screened using the PEGRx HT screening kit from Hampton

research as well as the Classics suite, and AmS04 suite kits from Qiagen. Wells

contained 2 μL of crystallization solution combined with 2 μL of 20 mg/mL GST-OYE 3

fusion protein in 50 mM Tris-Cl, 50 mM NaCl buffer pH 7.5 and used sitting drop vapor

diffusion. The best crystals obtained were quasi crystals obtained from well G4 of

PEGRx HT screening kit. These crystals were grown for 18 days with 5 mg/mL GST-

OYE 3 in 0.1 M Citrate, 20% (w/v) PEG 4000, 18% isopropanol, at pH 5.5 at room

temperature.

Native OYE 3 Protein Purification and Crystallogenesis

E. coli BL21 (DE3) Gold harboring plasmid pRP4 (a derivative of pET22b with an

OYE 3 coding region flanked by NdeI and XhoI sites) was grown at 37°C in a 4 L

fermenter containing LB medium supplemented with 200 μg/mL ampicillin (LB-amp).

Cells were grown in the fermenter with 600 rpm of agitation for 2 h to achieve mid log

151

phase, then induced by adding IPTG to a final concentration of 0.4 mM and grown with

100 mM glucose at 30°C with 600 rpm of agitation for 3 h. The cell solution was then

chilled at 4°C for 30 min before centrifugation at 5,000 × g. The wet cell pellet was then

resuspended in 1 mL of 100 mM Tris-Cl, pH 8.0 containing 10 μM PMSF per gram of

wet cell pellet. Cells were then lysed under 12,000 psi with the aid of a French Press.

The lysate was centrifuged for 1 h at 15,000 × g. Nucleotides were precipitated out of

supernatant by adding protamine sulfate to a final concentration of 1 mg/mL and stirring

for 20 min at 4°C. The supernatant was separated by centrifugation at 15,000 × g for 20

min. Proteins were precipitated by adding 5 portions of solid ammonium sulfate every 5

min to achieve a concentration of 78% saturation. The pellet was recovered by

centrifuging at 15,000 × g for 1 hr.

The ammonium sulfate pellet was resuspended in 100 mM Tris-Cl, 100 mM

(NH4)2SO4, 10 μM PMSF, pH 8.0, then dialyzed against 1 L of this buffer overnight at

4ºC. The following day the sample was then dialyzed for 2 h against 1 L of the same

buffer containing 10 mM sodium dithionite. After 2 h the buffer was exchanged for a

fresh 1 L of buffer containing 10 mM sodium dithionite and dialysis continued for 2 h.

The sample was then transferred to a fresh 1 L of buffer without dithionite and dialyzed

for 2 h, after which, the buffer was exchanged with a final 1 L of fresh buffer and

dialyzed overnight. The final sample was then centrifuged at 15,000 g for 30 min to

remove any insoluble debris accumulated during dialysis, then it was applied to a N-(4-

hydroxybenzoyl) aminohexyl agarose affinity column with the aid of an FPLC.

The affinity column was packed with 3 mL of the matrix and was equilibrated with

100 mM Tris-Cl, 100 mM (NH4)2SO4, 10 μM PMSF buffer at pH 8.0, prior to use. The

152

flow rate during purification was 0.5 mL/min. Elution of non-bound protein was

monitored by absorbance at 280 nm and a baseline established prior to loading protein

sample onto the column. Dialyzed protein samples were loaded onto the affinity column

in 10 mL portions. OYE 3 binding to the column gave a green color with a slightly bluer

tint than observed for OYE 1. Bound OYE 3 was washed with starting buffer until 280

nm absorbance returned to the baseline, this required anywhere between 30-60 mL of

buffer. The OYE 3 was eluted by washing with 100 mM Tris-Cl, 100 mM (NH4)2SO4, 10

μM PMSF, 4 mM sodium dithionite, pH 8.0. The collected protein sample was further

purified by gel filtration with a Superdex 200 column (Pharmacia) using 50 mM Tris-Cl,

50 mM NaCl, pH 7.5 at a flow rate of 0.5 mL/min. Fractions containing OYE 3 were

combined and concentrated by ultrafiltration to a final concentration of 40 mg/mL using

an Amicon tube. Protein concentration was determined by absorbance at 280 nm using

an extinction coefficient (ε) and molecular weights (MW) estimated by protparam (OYE

3 had an ε of 76,905 M-1cm-1 with a MW of 44,920 Da).

Crystallization conditions were screened using the PEGRx HT screening kit from

Hampton research as well as the Classics and AmS04 suite kits from Qiagen. Wells

contained 2 μL of crystallization solution combined with 2 μL of 20 mg/mL OYE 3 in 50

mM Tris-Cl, 50 mM NaCl, pH 7.5 and used sitting drop vapor diffusion. The best crystals

were obtained after 14 days using 100 mM MES monohydrate, 22% (v/v) PEG 400, pH

6.0 (well A8 of the PEG RX screening kit) at room temperature. No further optimization

of these crystallization conditions was required. Crystals were soaked in a harvesting

buffer for 5 s (100 mM MES monohydrate, 22% v/v PEG 400, 15% (v/v) glycerol, pH

6.0) before being flash cooled in liquid nitrogen and sent for synchrotron data collection.

153

OYE 3 with bound p-HBA was crystallized using the PEGRx HT screening kit

from Hampton research. Wells contained 2 μL of crystallization solution combined with 2

μL of 16 mg/mL OYE 3 in 50 mM Tris-Cl, 50 mM NaCl, pH 7.5 and used sitting drop

vapor diffusion. The best crystals were obtained after 10 days using 100 mM sodium

citrate tribasic dihydrate, 30% (v/v) polyethylene glycol monomethyl ether 550, pH 5.0

(well A10 of the PEG RX screening kit) at room temperature. The ligand soaking

occurred during harvesting. Crystals were soaked in harvesting buffer containing 2 mM

p-HBA for 5 s (100 mM Sodium citrate tribasic dihydrate, 30% (v/v) polyethylene glycol

monomethyl ether 550, 2 mM p-HBA, 15% (v/v) glycerol, pH 5.0) before being flash

cooled in liquid nitrogen and sent to the synchrotron for data collection.

OYE 3 W116V was crystallized using a PEGRx HT screening kit from Hampton

research. Wells contained 2 μL of crystallization solution combined with 2 μL of 27

mg/mL OYE 3 in 50 mM Tris-Cl, 50 mM NaCl, pH 7.5 and used sitting drop vapor

diffusion. The best crystals were obtained after 30 days using 100 mM sodium citrate

tribasic dihydrate, 30% (v/v) polyethylene glycol monomethyl ether 550, pH 5.0 (well

A10 of the PEG RX screening kit) at room temperature. The ligand soaking occurred

during harvesting. Crystals were soaked in harvesting buffer containing 2 mM p-HBA for

5 s (100 mM Sodium citrate tribasic dihydrate, 30% (v/v) polyethylene glycol

monomethyl ether 550, 2 mM p-HBA, 15% (v/v) glycerol, pH 5.0) before being flash

cooled in liquid nitrogen and sent for synchrotron data collection. Ligand soaking was

unsuccessful.

OYE 1 F296S was crystallized using the published conditions.20 Wells contained

6 μL of crystallization solution combined with 6 μL of 20 mg/mL OYE 1 F296 in 50 mM

154

Tris-Cl, 50 mM NaCl, pH 7.5 and used hanging drop vapor diffusion. The best crystals

were obtained after 7 days using 35% (v/v) PEG 400, 100 mM Na HEPES, 200 mM

MgCl2 buffer pH 8.3 at 6°C. The ligand soaking occurred during harvesting. Crystals

were soaked in harvesting buffer containing 2 mM p-HBA for 5 s (35% (v/v) PEG 400,

100 mM Na HEPES, 200 mM MgCl2, 2 mM p-HBA, 15% (v/v) glycerol, pH 8.3) before

being flash cooled in liquid nitrogen and sent for synchrotron data collection. Ligand

soaking was unsuccessful.

Alkene Substrates for OYE 3

A list of substrates and products is shown if Figure 4-6 through 4-8.








D). 2-(Hydroxymethyl)-cyclopent-2-enone eluted near 13.1 min. The reduced products

(S)- and (R)-6 eluted near 11.4 and 10.2 min, respectively.







155


D). 2-(Hydroxymethyl)-cyclohex-2-enone eluted near 13.1 min. The reduced products

(S)- and (R)-7 eluted near 10.2 and 10.8 min, respectively.

(R)-Pulegone (3)

(R)-Pulegone was purchased from Sigma Aldrich and can be detected during

screening by GC-MS using a DB-17 column (0.25 mm x 30 m). The temperature

program began with an initial temperature of 90°C, followed by an increase at 10°C/min

to a temperature of 130°C, followed by an increase at 2°C/min to a temperature of

150°C, followed by an increase at a rate of 20°C/min to a temperature of 250°C, at

which the program remained for 5 min (GC method is listed as YAP.Meth in Appendix

D). (R)-Pulegone eluted near 8.44 min and the reduced products cis- and trans-8 elute

at 6.01 min and 6.37 min (unassigned).

S-(+)-Carvone (4)

S-(+)-Carvone was purchased from Sigma Aldrich and it can be detected by GC-

MS using a DB-17 column (0.25 mm x 30 m). The temperature program began with an

initial temperature of 90°C, followed by an increase at 10°C/min to a temperature of

130°C, followed by an increase at 2°C/min to a temperature of 150°C, followed by an

increase at a rate of 20°C/min to a temperature of 250°C, at which the program

remained for 5 minutes (GC method is listed as YAP.Meth in Appendix D). S-(+)-

Carvone eluted near 8.8 min. A mixture of reduced product isomers, (+)-Dihydrocarvone

(Acros) was used as a standard to assign the peaks for both cis- and trans-9 (7.6 and

7.2 min, respectively).

156

R-(-)-Carvone (5)

R-(-)-Carvone was from Sigma Aldrich and can be detected by GC-MS using a

DB-17 column (0.25 mm x 30 m). The temperature program began with an initial

temperature of 90°C, followed by an increase at 10°C/min to a temperature of 130°C,

followed by an increase at 2°C/min to a temperature of 150°C, followed by an increase

at a rate of 20°C/min to a temperature of 250°C, at which the program remained for 5

min (GC method is listed as YAP.Meth in Appendix D). R-(-)-Carvone eluted near 8.8

min. A mixture reduced product isomers, (+)-Dihydrocarvone (Acros) was used as a

standard to assign the peaks for both cis- and trans-10 (7.6 and 7.2 min, respectively).


2-Methyl-2-cyclopenten-1-one was from Sigma Aldrich and can be detected

during by GC-FID using a Beta Dex 225 column (0.25 mm x 30 m). The temperature

program began with an initial temperature of 100°C for 10 min, followed by an increase

at 20°C/min to a temperature of 180°C, at which the program remained for 3 min. (GC

method is listed as BTS2.Meth in Appendix D). 2-Methyl-2-cyclopenten-1-one eluted

near 9.1 min and the reduced products (S)- and (R)-16 eluted near 5.9 min and 5.7 min

(unassigned).

2-Methyl-2-cyclohexen-1-one (12)

2-Methyl-2-cyclohexen-1-one was from Sigma Aldrich and was detected by GC-

MS using a DB-17 column (0.25 mm x 30 m). The temperature program began with an

initial temperature of 60°C for 2 min, followed by an increase at 10°C/min to a

temperature of 195°C, at which the program remained for 10 min (GC method is listed

as JON.Meth in Appendix D). 2-Methyl-2-cyclohexen-1-one eluted near 8.5 min and the

reduced product 2-methylcyclohexanone (Sigma Aldrich) eluted near 7.2 min.

157

3-Methyl-cyclohexen-1-one (13)

3-Methyl-cyclohexen-1-one was commercially available and can be detected by

GC-FID using a Beta Dex 225 column (0.25 mm x 30 m). The temperature program

began with an initial temperature of 70°C for 2 min, followed by an increase at

0.3°C/min to a temperature of 90°C which then immediately increased at 20°C/min to

temperature of 180°C, at which the program remained for 3 min. (GC method is listed

as BTS4.Meth in Appendix D). 3-Methyl-cyclohexen-1-one eluted near 73.1 min and the

reduction products (S)- and (R)-18 eluted near 47.4 and 48.2 min, respectively.

3-Ethyl-cyclohexen-1-one (14)

3-Ethyl-cyclohexen-1-one was prepared by in our lab by Magdalena Swiderska

using the method developed by Chandrasekhar and Reddy.12,86 3-Ethyl-cyclohexen-1-

one can be detected by GC-FID using a Beta Dex 225 column (0.25 mm x 30 m). The

temperature program began with an initial temperature of 70°C for 2 min, followed by an

increase at 0.3°C/min to a temperature of 90°C which then immediately increased at

20°C/min to temperature of 180°C, at which the program remained for 3 min. (GC

method is listed as BTS4.Meth in Appendix D). 3-Ethyl-cyclohexen-1-one eluted near

73.5 min and the reduction products (S)- and (R)-19 eluted near 59.7 and 62.2 min,

respectively.

3-Methyl-cyclopenten-1-one (15)

3-Methyl-cyclopenten-1-one was from Sigma Aldrich and can be detected during

screening by GC-FID using a Beta Dex 225 column (0.25 mm x 30 m). The temperature

program began with an initial temperature of 90°C for 20 min, followed by an increase at

20°C/min to a temperature of 180°C, at which the program remained for 3 min. (GC

method is listed as BTS3.Meth in Appendix D). 3-Methyl-cyclopenten-1-one eluted near

158

24.9 min and the reduction products (S)- and (R)-20 eluted near 15.2 and 15.7 min,

respectively.

4-Ethyl-4-methyl-2-cyclohexen-1-one (21)

4-Ethyl-4-methyl-2-cyclohexen-1-one was prepared in our lab by Bradford

Sullivan using the method developed by Flaugh et al.58 4-Ethyl-4-methyl-2-cyclohexen-

1-one can be detected by GC-FID using a Beta Dex 225 column (0.25 mm x 30 m). The

temperature program began with an initial temperature of 100°C for 30 min, followed by

an increase at 20°C/min to a temperature of 180°C, at which the program remained for

3 min. (GC method is listed as BTS7.Meth in Appendix D). 4-Ethyl-4-methyl-2-

cyclohexen-1-one eluted near 29.5 min and the reduced product 26 eluted near 21.4

min.

4-Isopropyl-4-methyl-2-cyclohexen-1-one (22)

4-Isopropyl-4-methyl-2-cyclohexen-1-one was prepared in our lab by Bradford

Sullivan using the method developed by Flaugh et al.58 4-Isopropyl-4-methyl-2-

cyclohexen-1-one can be detected by GC-FID using a Beta Dex 225 column (0.25 mm x

30 m). The temperature program began with an initial temperature of 100°C for 30 min,

followed by an increase at 20°C/min to a temperature of 180°C, at which the program

remained for 3 min. (GC method is listed as BTS7.Meth in Appendix D). 4-Isopropyl-4-

methyl-2-cyclohexen-1-one eluted near 33.4 min and the reduced product 27 eluted

near 31.6 min.

4,4-Diethyl-2-cyclohexen-1-one (23)

4,4-Diethyl-2-cyclohexen-1-one was prepared in our lab by Bradford Sullivan

using the method developed by Flaugh et al.58 4,4-Diethyl-2-cyclohexen-1-one can be

detected by GC-FID using a Beta Dex 225 column (0.25 mm x 30 m). The temperature

159

program began with an initial temperature of 100°C for 15 min, followed by an increase

at 20°C/min to a temperature of 195°C, at which the program remained for 10 min. (GC

method is listed as BTS8.Meth in Appendix D). 4,4-Diethyl-2-cyclohexen-1-one eluted

near 20.1 min and the reduced product 28 eluted near 19.7 min.

Spiro[5.5]undec-1-en-3-one (24)

Spiro[5.5]undec-1-en-3-one was prepared in our lab by Bradford Sullivan using

the method developed in by Flaugh et al.58 Spiro[5.5]undec-1-en-3-one can be detected

by GC-FID using a Beta Dex 225 column (0.25 mm x 30 m). The temperature program

began with an initial temperature of 100°C for 15 min, followed by an increase at

20°C/min to a temperature of 195°C, at which the program remained for 10 min. (GC

method is listed as BTS8.Meth in Appendix D). Spiro[5.5]undec-1-en-3-one eluted near

23.4 min and the reduced product 29 eluted near 22.7 min.

2-Butylidenecyclohexanone (25)

2-Butylidenecyclohexanone was prepared in our lab by Magdalena Swiderska

using a method developed by Huang et al.12,87 2-Butylidenecyclohexanone can be

detected by GC-FID using a Beta Dex 225 column (0.25 mm x 30 m). The temperature

program began with an initial temperature of 70°C with an increase at 0.3°C/min to a

temperature of 100°C which then immediately increased at 20°C/min to temperature of

190°C, at which the program remained for 3 min. (GC method is listed as BTS10.Meth

in Appendix D). 2-Butylidenecyclohexanone eluted near 93.0 min and (S)- and (R)-30

eluted near 47.4 and 48.2 min, respectively.

Screening

E. coli BL21 (DE3) Gold cells harboring plasmids encoding wild-type and W116

site-saturation OYE 3 mutants were grown in a 96 well plate containing 600 μL of LB-

160

amp. Cells were grown at 37°C with 250 rpm of agitation overnight. The saturated

cultures were then used to inoculate a larger 2 mL square bottom 96 well plate. This

larger square bottom plate contained 600 μL of an auto-induction medium. The auto-

induction medium contained a mix of ZY media, 50x5052, 20x NPS, and 200 μg/mL

ampicillin.33 Cells were induced in an aeration case developed at the University of

Florida by the machine shop in the Department of Chemistry. Induction occurred at

37°C with 350 rpm of agitation overnight. The increased agitation is required for

induction to occur. Induced cells were then separated from the auto-induction medium

by centrifugation. Auto-induction medium was removed and induced pelleted cells were

then resuspended in 600 μL of a reaction mixture. The reaction mixtures contained 50

mM KPi buffer with 100 mM glucose and 15 mM of the substrate of interest at pH 7.0.

Reactions were run at room temperature with 250 rpm of agitation overnight. Reactions

were quenched by adding 500 μL of ethyl acetate. The organic phase was separated by


Conclusions

We discovered multiple crystallization conditions for wild-type OYE 3 and OYE 3

W116 mutants and we also solved the structure of OYE 3. Given the sequence identity

between OYE 3 and OYE 1 (80%), it is not surprising that these homologs also have

very similar structures. The only significant difference in the active site is the presence

of a serine at position 296 on loop 6 in OYE 3 (OYE 1 has a phenylalanine at position

296). Our results have highlighted the importance of this active site residue, which will

be the subject of future studies. There is significant potential for protein engineering of

both OYE 1 and OYE 3 within loop 6. Whether variants with a combination of mutations

at W116 along with loop 6 mutations at position 296 can provide synergy to open up

161

OYE homologs to new substrates is yet to be seen; however, we now have a complete

set of OYE 3 W116 site-saturation mutants to survey and the means to investigate their

structures.

With regard to the substrate profiling of OYE 3 and site-saturation mutants for

W116, we learned that OYE 3 retains its preference for binding both carvone

enantiomers in the “normal” substrate binding mode, regardless of the nature of the

residue at position 116. This stands in stark contrast to the case for the analogous OYE

1 W116 mutants, many of which gave “flipped” substrate binding. For example, OYE 1

the A, C, E, G, I, M, N, Q, S, T and V replacements for W116 all afforded trans-9, the

result of “flipped” substrate binding.24 The same mutations in OYE 3 mutants gave no

trans-9, and only the “normal”, cis-9 product was observed. For (R)-carvone, both the

OYE 1 W116A and W116V variants give the product of flipped binding (cis-10). The

corresponding mutants in OYE 3 provide a 1 : 1 mixture of diastereomers. One other

difference in stereopreference was observed for substrates 1 and 2. The W116E mutant

of OYE 1 yielded (S)-6 and (S)-7 with ca. 90% ee. The W116E mutant of OYE 3 gave

racemic mixtures of 6 and 7. Finding such variations between these very similar OYE

homologs is fortunate and suggests that novel variants with even more different

stereoselectivities may be accessible after further study of OYE 3.

162

Table 4-1. Crystallographic data collection and refinement statistics.

Structure title OYE 3 Wt OYE 3 W116V OYE 3 soaked in p-HBA

Ligand Soaked none none p-HBA

X-Ray Source 21-ID-G beamline 21-ID-G beamline 21-ID-G beamline

APS Argonne National

Laboratory

APS Argonne

National Laboratory


Laboratory

Space Group P 21 21 21 P 21 21 21 P 21 21 21


61.214 107.762 141.071

90 90 90

61.63 106.935 140.46

90 90 90

61.63 106.421 141.01 90

90 90

a, b, c (Å)

Resolution (Å) 38.88 - 1.798

(1.862 - 1.798)

24.98 - 1.88

(1.947 - 1.88)

24.92 - 1.88

(1.947 - 1.88)

Unique Reflections 87271 (8450) 76198 (7496) 76002 (7479)

Completeness (%) 1.00 (0.98) 0.99 (1.00) 1.00 (1.00)

Multiplicity 14.5 (14.3) 14.3 (14.8) 14.314.6 (14.7)

Rsym 0.09911 (0.6901) 0.1248 (0.5961) 0.122 (0.5929)

I/σ (I) 20.18 (3.95) 15.01 (4.25) 18.53 (5.34)

Rwork 0.1581 (0.2360) 0.1999 (0.2362) 0.1820 (0.2073)

Rfree 0.1916 (0.2684) 0.2392 (0.2956) 0.2185 (0.2536)

Ramachandran

Statistics

Favored (%) 97 97 97

Allowed (%) 2.7 2.9 3.2

Outliers (%) 0 0 0

Number of Protein,

Solvent, and Ligand

Atoms

6309, 795, 64 6261, 790, 62 6325, 792, 126

Average B Factors

(Å2) 20.78 22.52 18.53

Protein 20.14 22.34 18.16

Ligands 18.65 17.14 25.12

Solvent 26.96 26.42 22.35

Values in parentheses denote data for the highest resolution bin (1.862 - 1.798 Å, 1.947 - 1.88 Å, and 1.947 - 1.88 Å)

163

Table 4-2. Crystallographic data collection and refinement statistics.

Structure title OYE 1 F296S

Ligand Soaked none

X-Ray Source 21-ID-G beamline


Laboratory

Space Group P 43 21 2


141.5 141.5 42.54

90 90 90

a, b, c (Å)

Resolution (Å) 24.98 - 1.88

(1.92 - 1.85)

Unique Reflections 37426 (3651)

Completeness (%) 1.00 (0.99)

Multiplicity 3.8 (3.8)

Rsym 0.0587 (0.669)

I/σ (I) 19 (2.6)

Rwork 0.1843 (0.2756)

Rfree 0.2225 (0.3390)

Ramachandran

Statistics

Favored (%) 96

Allowed (%) 3.9

Outliers (%) 1.8

Number of Protein,

Solvent, and Ligand

Atoms

3172, 410, 32

Average B Factors

(Å2) 26.52

Protein 25.25

Ligands 19.58

Solvent 36.86

Values in parentheses denote data for the highest resolution bin (1.92 -1.85 Å).

164

Table 4-3. Distances between the β-carbon of each active site residue to the ligand.

OYE 1 Residue

β-carbon Distance (Å)

Ref. Carbon # OYE 3 Residue

β-carbon Distance (Å)

N194 4.1 1 N194 4.2

Y196 6.7 1 Y196 6.8

G72* 6.4 2 G72 6.3

W116 7.0 2 W116 7.6

L118 8.9 2 L118 9.2

H191 7.3, (7.4) 2, (1)** H191 7.7, (7.5)**

T37 4.0 3 T37 3.8

M39 9.4 3 M39 9.4

Y82 9.0 3 Y82 9.2

F374 9.1 4 F374 8.9

Y375 9.1 4 Y375 9.3

N294 9.4 5 D294 8.9

P295 5.1 5 P295 4.8

F296 7.8 5 S296 7.4

F249 6.8 6 F250 6.4

N250 8.3 6 N251 7.9

Distances are measured in angstroms from the β-carbon of the indicated residue to the nearest ring carbon of the bound ligand. Phenolic carbon is designated as 1 and proceeds clockwise as viewed from above the bound FMN cofactor.28 *Measurement taken from α-carbon **Position H191 in OYE 3 is closer to C1 than the C2, which is reverse in OYE 1.

165

Figure 4-1. Schematic illustration of the FMN environment in the active site of OYE homologs. The FMN (Gold) environment of OYE 1 (green), OYE 2.6 (violet), and OYE 3 (magenta). OYE 3 uses the same hydrogen bonding partners as OYE 1 to lock the FMN into place within the active site (T37, G72, Q114, and R243). OYE 3 also uses the same hydrophobic partners beneath the FMN and shown with blue circles (P35, L36, and I351) to lock it into position.

Figure 4-2. The mechanism of OYE 3. Mechanism for the reduction of a bound α-unsaturated carbonyl substrate (black) by a reduced FMN (gold) in the active site of OYE 3 (magenta). OYE 3 uses the same hydrogen bonding partners as OYE 1 (H191 and N194) to lock the carbonyl into position.

166

Figure 4-3. Diagram of the positions in the active site of OYE homologs. The active site positions of OYE 1 (green), OYE 2.6 (violet), and OYE 3 (magenta). All positions except S296 in OYE 3 have the same residues as in OYE 1.

167

Figure 4-4. Loop 6 in OYE homologs. This figure shows loop 6 in OYE 1 (green) and OYE 3 (magenta) along with FMN (yellow) and a bound p-HBA ligand. This portion of the active site has the most variability across the structures of OYE homologs. The loop 6 of OYE 3 allows for the largest amount of unoccupied space south of the bound substrate.

168

Hall et al. (2008)42

Stueckler et al. (May 2010)14

Figure 4-5. List of OYE 3 substrates and reported conversion from the literature.

169

Stueckler et al. (October 2010)44

Stueckler et al. (2011)88


170

Brenna et al. (June 2011)45



171

Brenna et al. (December 2011)47

Tasnadi et al. (March 2012)51


172

Tasnadi et al. (June 2012)50

Durchschein et al. (2012)49


173



Brenna et al. (February 2012)90


174


Knaus et al. (2014)91


175

Brenna et al. (March 2014)53



176

Turrini et al. (2015)55


177

Figure 4-6. First set of substrates and theoretical binding mode products.

178

Figure 4-7. Second set of substrates and theoretical binding mode products.

179

Figure 4-8. Third set of substrates and theoretical binding mode products.

180

Figure 4-9. The reactions used to test phenol binding by OYE 3. Substrate 12 was used with and without 4-chlorophenol.

Figure 4-10. Crystals and crystallization conditions for of OYE 1 (left), OYE 2.6 (middle), and OYE 3 (right).

181

Figure 4-11. The structure of OYE 3. The OYE 3 structure has an alpha beta barrel with the FMN (Yellow) located within the barrel.

Figure 4-12. The active site for both OYE 1 (green) and OYE 3 (magenta) with bound FMN (yellow) and substrate (OYE 3 blue and OYE 1 green).

182

Figure 4-13. The active site for both OYE 3 (magenta) and OYE 3 W116V (orange) with bound FMN and p-HBA (cyan).

Figure 4-14. The active site for both OYE 1 (green) and OYE 1 F296S (orange) with bound FMN and p-HBA (cyan).

183

Figure 4-15. Results from screening the OYE 3 W116 site-saturation library against substrate 1.


184



185



186



187



188



189



190

CHAPTER 5 IMPROVING THE SUBSTRATE RANGE OF AMINOLEVULINIC ACID SYNTHASE


Background

Enzymes that utilize pyridoxal phosphate (PLP) cofactors can perform an

impressive set of reactions. 5-Aminolevulinic acid synthase (ALAS, EC 2.3.1.37) is a

PLP dependent enzyme that performs a Claisen-like condensation between succinyl-

CoA and glycine to produce 5-aminolevulinic acid (δ-AL, ALA) (Figure 5-1).92–98 Several

studies on the kinetic mechanism of ALAS have been completed and have established

a proposed pathway by which glycine and succinyl-CoA combine to form δ-AL.99–102 In

the proposed mechanism (Figure 5-2), the decarboxylation of the bound glycine occurs

only after the Claisen condensation step and thus, may not be essential for catalysis.

We believe that this implies that there is potential for incorporating other amines that

lack carboxylate moieties as substitutes for glycine, which would significantly extend the

synthetic utility of ALAS. In support of this notion, it has been shown that methyl amine

binds to PLP and forms the external aldimine in ALAS.103 Formation of the external

aldimine by the binding of a substrate with PLP produces an increase in absorbance at

420 nm. The Ferreira group was able to prove that a methyl amine substrate would bind

to PLP and form the external aldimine by monitoring A420 absorbance. Whether other

amines can form the external aldimine and / or complete the catalytic cycle of ALAS

remains to be determined. All of these results taken together make ALAS an excellent

candidate for synthetic applications and protein engineering. Below, several positions

that have been subjected to site-directed mutagenesis are reviewed.

191

Positions of Interest

Threonine 148

ALAS has been shown to be receptive towards protein engineering and mutants

that improved its substrate range and enhanced its catalytic efficiency have been

reported.104 Protein engineering of murine erythroid-specific ALAS (mALAS) has

revealed that mutations at position T148 can allow the enzyme to accept alternate

amino acids as substrates.100 Threonine at position 148 is conserved amongst the

structures of ALAS and the crystal structure of R. capsulatus ALAS shows that the

threonine at this position is located on a loop that directs its side-chain into the active

site (Figure 5-3).105 Furthermore, this side-chain lies near the glycine bound to PLP. The

steric bulk of the threonine side-chain at position 148 would make it difficult for any

substrate other than glycine to bind to PLP. Wild-type ALAS can accept serine in place

of glycine as a substrate, although the conversion is very poor. Mutagenesis studies

targeting T148 were carried out to improve the capacity of ALAS to accept serine.100 An

ALAS T148A mutant better accommodated serine, presumably because of the small

side-chain volume at this position. These results suggested to us that additional

mutations at position 148 might prove fruitful in further extending the substrate range of

ALAS.

Isoleucine 151

Isoleucine 151 is a second candidate for mutagenesis. The R. capsulatus ALAS

crystal structure shows that the side-chain of I151 also extends off the same loop as

T148 into the ALAS active site. Similar to T148, an isoleucine at position 151 is

generally conserved within the sequence of ALAS homologs.106 We hypothesized that

the side-chain of I151 partially dictates the position of the T148 side-chain. This would

192

mean that residues at both positions 148 and 151 cooperatively impact amine substrate

specificity. For this reason, we chose position 151 for mutagenesis.

Arginine 85

ALAS accepts both β-hydroxybutyryl-CoA or acetoacetyl-CoA as substitutes for

succinyl-CoA, albeit at reduced relative rates (32% and 13%, respectively).100 Altering

R85 altered these substrate preferences and expanded the range of usable acyl-CoAs.

Ferreira mutated R21 in R. capsulatus ALAS (equivalent to R85 in murine ALAS) to

leucine and lysine This changed the enzyme’s preference from succinyl-CoA to butyryl-

and octanoyl-CoA.99 This result shows there is the potential to engineer this enzyme to

not only accept substitutes for the amino acid substrate (glycine), but also substitutes

for the acyl-CoA substrate (succinyl-CoA). This makes position R85 yet another

excellent candidate for mutagenesis.

The Glycine Loop

Amino acids T148 and I151 are located on an active site loop that directs both

residues into the proximity of the bound glycine external aldimine complex. Both of

these positions are of high interest for protein engineering. We intended to carry the

previous studies further and make full degenerate libraries at both positions T148 and

I151. Similarly, substitutions at position R85 have produced mALAS mutants that will

accept acyl compounds like butyryl-CoA and octanoyl-CoA as substitutes for succinyl-

CoA.99 We believe that this made position R85 another excellent position for

mutagenesis. Lastly, the loop that directs positions T148 and I151 is an area of interest

we wished to explore through randomization. Modifications along this loop may alter it in

subtle ways that would open up the area near the Pro-S hydrogen of a bound glycine,

thereby allowing amino acids with additional α-carbon functionality to bind. In an effort to

193

effectively probe this region of the active site for interesting mutants, we wanted to

randomize the entire loop through cassette mutagenesis. Since the region of interest is

large, randomization in this manner would be the ideal way to tease out the mutants we

want.

Our long-term goal was to make ALAS mutants that could accept any amino acid

in addition to glycine and also accept a variety of acyl-CoA substrates. As ALAS

converts glycine and succinyl-CoA to δ-AL, it selectively deprotonates the Pro-R

hydrogen of glycine during a series of steps towards producing δ-AL. Since ALAS uses

a chirally-selective step in the mechanism, there is potential to use this enzyme to

produce chiral products (even though the normal product, δ-AL, is achiral). By

engineering ALAS to accept amino acid with chiral α-carbons, we hoped that the

products would maintain the optical purities of the reactants. Given the large range of

substrates to be explored, the significant extent of protein engineering planned, and the

laborious efforts required to purify mALAS mutants, we first required a “prescreening”

assay for our mutants that would reveal their catalytic (or lack of catalytic) activity

without requiring protein purification. Our plan was to prescreen transformants grown in

a δ-AL knockout host using the native reaction of glycine and succinyl-CoA to make δ-

AL, then further examine active variants with additional substrates using

spectrophotometric assays. The most promising transformants would be purified and

further characterized. This workflow was designed to cut down significantly the time

wasted by purifying nonfunctional mutants.

Project Overview

We assembled complete site-saturation mutagenesis libraries for each of the

three ALAS positions of greatest interest: R85, T148, and I151. We then prepared a

194

template to be used for cassette mutagenesis at the glycine loop between positions

141-156. The original pGF23 vector was prepared with cassette mutagenesis in mind

but this vector did not target the areas of this loop as exclusively as we intended.104,107 A

silent mutation was introduced at position 161 that placed an Eco53kI restriction site at

this location. This site flanked the loop residues of interest, along with a pre-existing

AleI. This greatly simplified cassette mutagenesis that simultaneously targeted multiple

positions within the glycine loop.

We developed a prescreening assay for ALAS catalytic activity by derivatizing

the δ-AL-pyrrole with Ehrlich’s reagent. By reacting δ-AL with acetylacetone, a δ-AL-

pyrrole is made which is amenable to derivatization with Ehrlich’s reagent (Figure 5-4)

that forms an adduct with very strong absorbance at 553 nm (ϵ = 68,000 cm-1M-1) from

which the δ-AL concentration can be measured.108–110 Since this assay is robust and

can be used on a 96 well plate, it was ideal for screening several mutants.

We next developed an assay for measuring catalytic activity of mutants identified

from the prescreening step. One strategy was to quantitate acyl-CoA species using

reversed phase HPLC111 This would allow us to monitor our reactions that use acyl-CoA

substrates from the loss of these species and the appearance of free CoASH. We

validated this method by separating free CoASH from succinyl-CoA using HPLC (Figure

5-5).112,113 The advantage of this assay was that a variety of different acyl-CoA species

(not just succinyl-CoA) could be accommodated. The amino donor could also be varied

beyond glycine in this methodology. The disadvantage was that any acyl-CoA cleavage,

e.g., by spontaneous hydrolysis, added a background rate of acyl-CoA consumption.

195

The second strategy for measuring ALAS catalytic activity was a coupled

spectrophotometric assay that used α-ketoglutarate dehydrogenase (α-KGD) to produce

succinyl-CoA in situ from CoA and α-ketoglutarate in an NAD+-dependent reaction. This

allowed the use of catalytic amounts of CoASH in the reactions and allowed the reaction

progress to be followed by measuring NADH formation by UV-Vis (Figure 5-6).114 This

assay could be used for amino substrates other than glycine, although it was limited to

succinyl-CoA as the second reactant.

The final analytical method we attempted to develop directly measured formation

of the final product of ALAS. We originally explored the nitrogen-derivatization reagent

phenylisothiocyanate (PITC, Edman’s reagent) prior to HPLC analysis of the reaction

mixture (Figure 5-7).115,116 Unfortunately, this reagent proved unsuccessful for δ-AL, so

we explored other methods based on GC-MS and MSTFA (Figure 5-8).117 Optimizing

this protocol to work with ALAS reactions would provide the final analytical tool to

evaluate ALAS mutants with either acyl-CoA or amino acid substitutes.


Detecting δ-AL-Pyrrole Compounds with Ehrlich’s Reagent

Our first task was to develop an assay that could detect δ-AL and other amino

products. The reaction of δ-AL with acetyl acetone produces a δ-AL-pyrrole. This

pyrrole will form a δ-AL-pyrrole Ehrlich derivative if subsequently reacted with Ehrlich’s

reagent. The resulting Ehrlich derivative is detectable at A553, with a ϵ = 68,000 cm-1M-1,

which allows quantification of the δ-AL produced in reactions with ALAS. We were able

to use this reaction successfully on products from reactions using ALAS to make δ-AL

from glycine and succinyl-CoA (Figure 5-9). This reaction was also successful when

using a plate assay containing several redundant reactions of δ-AL (Figure 5-10 and 5-

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11). It was our hope to develop a mid-level screening assay using this reaction to

screen through samples that succeeded during the E. coli HU 227 cell screening.

Preparation and Detection of Succinyl-CoA

Next, we set out to synthesize and detect succinyl-CoA. The standard method

involves mixing succinic anhydride and free CoASH with minimal stirring in an ice bath

for 30 min.112,113 Both succinyl-CoA product and residual CoASH can be separated and

quantitated by HPLC analysis. This provided us with a means to monitor the reaction

and ensure that it proceeded to completion.111 Further evidence that our synthesis of

succinyl-CoA was successful was obtained by reacting the reaction product with excess

hydroxylamine (Figure 5-12). Hydroxylamine is a strong nucleophile that cleaves

thioesters, yielding thiols and hydroxamic acids.118,119 Here, hydroxylamine formed

succinyl hydroxamic acid and regenerated CoASH, which could be followed by HPLC

(Figure 5-13 through 5-15). Iron(III)chloride was added to product to detect the

hydroxamic acid co-product, the solution’s color changed from yellow to dark red

indicating success. No spectral experiments were done on the product.

In Situ Succinyl-CoA Formation

While equimolar quantities of succinyl-CoA could be used for our studies, a

system requiring only catalytic quantities of CoASH would be much less expensive

when applied to a large number of samples. We therefore utilized an in situ succinyl-

CoA regeneration system that uses α-Ketoglutarate Dehydrogenase (α-KGD) to make

succinyl-CoA from CoASH and α-ketoglutarate.120 This system requires molar

equivalents of NAD+, α-ketoglutarate, and CoASH to produce succinyl-CoA. However,

since free CoASH is released as a by-product of δ-AL production by ALAS, the ALAS

reaction can be coupled to that of the α-KGD and provide a renewable supply of

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succinyl-CoA using only a catalytic quantity of CoASH. The coupling of CoA production

to NADH production is also useful in that NADH can be detected and quantified by A340

(ϵ = 6,220 (M-1cm-1).120 Thus, this technique provided a method to indirectly quantify the

succinyl-CoA to CoA conversion using a simple spectrophotometric assay. In reactions

without ALAS, the production of NADH stopped at a level corresponding to the

concentration of added CoASH, which is the limiting reagent in the absence of ALAS.

After this time, NADH production occurred at a greatly reduced rate, probably due to

spontaneous hydrolysis of succinyl-CoA under the reaction conditions (Figure 5-16).

HPLC analysis demonstrated that succinyl-CoA from CoASH and α-ketoglutarate under

these reaction conditions.

We tested the utility of this spectrophotometric assay by examining purified wild-

type mALAS and a series of mutants, generously provided by the Ferreira group.104 The

succinyl-CoA regeneration system was allowed to incubate for 15 min in order to

produce an initial amount of succinyl-CoA. ALAS was then added and the conversion of

NAD+ into NADH was monitored by A340. This set of experiments proved that the

conversion of succinyl-CoA to CoASH could be monitored for ALAS reactions (Figure 5-

17). The usefulness of this coupled assay cannot be understated. Since in addition to

providing a replenishing source of succinyl-CoA, it also provided an indirect method to

test any substrate that would be substituted for glycine. This is crucial since it is was our

goal to test a sizeable number of substrates against a myriad of mutants and several of

those are amino acid substitutes.

Detecting Amino Products Using PITC and MSTFA Derivatives

We next set out to find an effective way to detect the reaction products that ALAS

might make when other amines were substituted for glycine. The spectrophotometric

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assay described above only detects succinyl-CoA consumption, rather than directly

indicating C-C bond formation. It is also limited to reactions using succinyl-CoA as a

partner. We therefore sought a direct method for product detection and quantitation. We

first tried the classical method for detecting amino acids: PITC derivatization and HPLC

separation.115,116 We derivatized a set of amino acid standards using PITC. These

standards included: phenylalanine, leucine, alanine, glutamate, glycine, and δ-AL.

Separation of the PIT-amino acid derivatives by HPLC worked well for all standards

except PIT-δ-AL (Table 5-1). This was very unfortunate. Though we would ultimately

make products other than δ-AL, the products we would make would be similar to δ-AL,

so if this assay did not work on the parent compound, another assay was needed.

We next tried using MSTFA derivatization to enable GC-MS identification and

quantitation of ALAS products.117 Since all of the potential ALAS products would have a

primary amine with a labile proton, we hoped that they would be receptive to N-silylation

using MSTFA. This was indeed the case for glycine and δ-AL (Figure 5-18).

Unfortunately, while this methodology worked well with amino standards dissolved in 50

mM HEPES, pH 7.5, it failed when KPi buffer at any concentration was employed, and

so HEPES buffer was used. Though the reaction worked well enough for standards in

buffer solution (only HEPES) it was never successful at working with reactions using the

enzyme or with the other reaction conditions like the regeneration system. Using

mALAS reaction conditions greatly increased the number of unidentified peaks on the

chromatogram which further complicated identifying, much less quantifying, the TMS-

glycine and TMS-δ-AL peaks. Also, the glycerol from the stock solutions of both mALAS

and α-KGD may have sequestered water, which would be released once the solution

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was dissolved in the MSTFA solution and interfere with the derivatization reaction. This

method was thus never successfully used on reactions with mALAS. Thus, this assay

requires a significant amount of further optimization before it can be used to reliably

quantify mALAS products.

mALAS R85, T148 and I151 Site-Saturation Mutagenesis Libraries

We completed the cloning of complete, individual site-saturation mutagenesis

libraries of mALAS at positions R85, T148, and I151. All mutants for these libraries were

made individually and then used to transform E. coli HU 277 (an ALAS knockout

strain).104 We found concatameric primer inserts from the PCR were in the sequencing

of several these mutants. The artifacts almost exclusively contained a single reverse

primer insert, rather than the random mix of primer inserts that we observed during the

sequencing of the OYE 2.6 Y78 mutants mentioned in Chapter 2. Also, concatameric

mutants did not occur nearly as often as they did in the OYE 2.6 Y78 mutations. The

ratio of successful mutants to concatameric failures was: 4:1 for R85, 1:3 for T148, and

1:1 for T151. Making and sequencing redundant mutants allowed us to overcome this

problem. However, given the laborious requirements of making so many redundant

samples for such a large number mutants, and the difficulties of optimizing all of the

analytical methods, the protein purification was not successfully optimized. And as such,

optimizing the protein purification protocol is the next necessary step to screen our

mutants.

Experimental

General



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from Integrated DNA Technologies. All other reagents were obtained from commercial

suppliers and used as received. Plasmids were purified on small scales by Wizard®

minicolumns (Promega Life Sciences) and on large scales using CsCl density gradient

ultracentrifugation. DNA sequencing was carried out by the University of Florida ICBR

using capillary fluorescence methods using standard protocols. LB medium contained 5

g/L Bacto-Yeast Extract, 10 g/L Bacto-Tryptone and 10 g/L NaCl.

Cloning

Construction of plasmid used to make ALAS libraries

The plasmid used as a template to make the ALAS libraries was pGF23 a CASS-

3 derivative prepared by Professor Gloria Ferreira, containing the gene for erythroid-

specific ALAS from mice (mALAS) (Appendix E, Figure E-5).107,121,122

Construction of ALAS libraries

The template used to make the mutants in all three ALAS libraries was pGF23. A

set of mutagenic primers containing a single codon replacement at either position R85,

T148, or I151 in ALAS was used to make each of the 19 mutants (primers are listed in

Appendix A, Table A-5). PCR was performed using 0.5 μL of 18 ng/μL template, 5 μL of

both 5 mM forward and reverse mutagenic primers, 1 μL of 10 mM dNTP mix, 10 μL of

5X HF Phusion® Hot start buffer, 28 μL of sterile water, and 0.5 μL of 2 U/μL Phusion®

Hot Start II DNA Polymerase for a total reaction volume of 50 μL. PCR was performed

using a MJ Mini® thermocycler from BioRad. PCR samples were run with an initial

denaturation step at 98°C for 30 s, then a subsequent 25 cycles of denaturation at 98°C

for 10 s, annealing at 64°C for 30 s, and an extension step at 72°C for 3 min 30 s, after

which the reactions were completed with a final extension step at 72°C for 7 min 30 s.

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with two doses of 0.5 μL of 20 U/μL DpnI at 37°C to remove the parent template. DpnI is

an endonuclease that targets hemi methylated DNA and as such is ideal for removing

the superfluous template. The first dose of DpnI was added immediately after PCR

clean up and the second was added after 4 h of digestion. After DpnI digestion, samples

were cleaned using Wizard® Plus SV Gel PCR Clean up kits by Promega, using the

manufacturer instructions.

Following the PCR work up, PCR samples were transformed by electroporation

into ETB cells using a Gene Pulser® from BioRad. Electroporation was carried out with

2 - 6 μL of PCR sample and 50 µL of ETB cells under 2.5 kV. Electroporated samples

were incubated in 600 μL of SOC media at 37°C for 1 h. Cells were then plated onto LB-

amp agar plates and grown at 37°C for 36 h. Transformant cells were sequenced to

verify that the desired mutation was present at the ICBR complex using Sanger

sequencing. Plasmids that were verified by sequencing to contain the desired mutant

were then transformed in an expression strain. Transformation was done using

electroporation under 2.5 kV with 4 μL of 10 ng/μL of plasmid with 80 μL of E. coli HU

227 electrocompetent cells. Electroporated samples were incubated in 600 μL of SOC

medium at 37°C for 45 min. Cells were then plated onto LB-amp agar plates and grown

at 37°C overnight. After mutations were verified by sequencing, transformants were

assembled into a 96 well plate. Transformants were grown in 600 μL of LB-amp in a 96

well plate overnight to reach saturation. The library was completed with the transfer of

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120 μL of saturated cultures into a new 96 well plate containing 30 μL of 80% glycerol

which brought the final concentration of glycerol to 15%.

Preparation and Detection of Succinyl-CoA

Succinyl-CoA was prepared using succinic anhydride and CoA. CoA was from

Sigma-Aldrich. Succinic anhydride was thoroughly ground using a beaker and wax

paper. An adequate amount (between 1/2 and 1/3 a centimeter) of ground succinic

anhydride was loaded into a yellow pipette tip by pressing into succinic anhydride.

Succinic anhydride was then transferred into a 100 μL aqueous solution containing 20

mM CoA. 800 μL of ice cold D.I. water was added to the solution bringing the CoA to a

concentration of 22 mM. The solution was then mixed every 5 min using a pipette while

in ice bath for 30 min. After 30 min 100 μL of 1 M HCO3 was added bringing solution to

final volume of 1 mL with 100 mM HCO3, and 2 mM succinyl-CoA. Succinyl-CoA was

immediately stored on ice and used for experiments.

Reactions were then verified by HPLC and separated on a 150 × 4.6 mm Synergi

Hydro-RP 80 Å column using reverse phase HPLC. Solvent A was 50 mM NaPi buffer

with a pH of 5.0 and Solvent B was 50 mM NaPi with a pH of 5.0 and 20 % of

acetonitrile. The flow rate was 1 mL/min and samples were monitored by UV

absorbance simultaneously at both 220 and 260 nm. Initial conditions (3% B) were

maintained for 2.5 min; then a linear increase to 18% B over 5 min was immediately

followed by a linear increase to 28% B over 3.5 min, then a linear increase to 90% B

over 10 min. After a 5 min hold at 90% B, a linear decrease to 3% B over 3 min was

followed by a 5 min hold at the initial conditions (3% B) (HPLC method is listed as

LMM.Meth in Appendix D).123 CoA had eluted at 14.9 min, succinyl-CoA eluted at 15.5

min, and caffeine eluted at 24.3 min (caffeine was used as an internal standard). The

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CoA peak was verified by spiking reactions with 20 mM CoA. A succinyl-CoA standard

was far too expensive to use, so an indirect method was needed to verify the succinyl-

CoA peak. The first method used to verify the succinyl-CoA was reaction with

hydroxylamine. Hydroxylamine can be used as a nucleophile to break thiol esters into

thiols and hydroxamic acids. In the case of this reaction, it displaces the succinyl adduct

forming succinyl-hydroxamic acid and regenerating CoA. By monitoring the

disappearance of the presumptive succinyl-CoA peak and the increase of the

established CoA peak on HPLC we were able to verify the succinyl-CoA peak’s position.

Further verification of the succinyl-CoA peak was done using the succinyl-CoA

regeneration system. Aliquots were taken from a reaction using the succinyl-CoA

regeneration system after the NADH production had topped off and analyzed by HPLC.

The major product peak from the succinic anhydride peak matched the major product

peak for the regeneration system.

Succinyl-CoA Regeneration System

A 2 mL reaction mix was prepared using 20 mM HEPES, 3.0 mM MgCl2,1.0 mM

α-ketoglutarate, 1.0 mM NAD+, 250 μM thiamine pyrophosphate, 100 mM amino acid

substrate, 20 μM CoA, and 2.9 U/mL α-KGD. 1 mL of this solution was aliquoted out to

be used as a blank and the other 1 mL was saved to be used for the reaction with

enzyme. Reactions were incubated at room temperature for 20 min to allow an initial

amount of succinyl-CoA to be made by α-KGD before being analyzed by

spectrophotometry. Reactions with enzyme included 10 µL of mALAS from a glycerol

stock solution (concentrations were not determined). Reactions were analyzed using a 2

mL quartz cuvette with a 1 cm path length in an Agilent 8453 UV-Vis spectrophotometer

at 30°C. Reactions were initiated by the addition of the mALAS. NADH can be detected

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and quantified by A340, with a ϵ = 6,220 (M cm)-1.120 Reactions were observed over a

course of 20 min. Aliquots of the reaction blank, solutions that were absent of any

mALAS, were separated by HPLC to verify that succinyl-CoA had been made.

Detecting δ-AL-Pyrrole Compounds with Ehrlich’s Reagent

2 mL of 0.2 mM δ-AL was combined with 5 mL of 1 M acetic acid buffer at pH

4.6. Then 200 μL of acetyl acetone was added to the solution. A sufficient quantity of

acetic acid buffer was added to bring the final volume to 10 mL with a concentration of

20 μM δ-AL. The reaction was then submerged in a boiling water bath for 10 min. 1 mL

aliquots of 20 μM, 10 μM, & 5 μM δ-AL-pyrrole were prepared from this stock solution

using 1 M acetic acid buffer to bring the volumes to 1 mL. Ehrlich’s reagent was

prepared by dissolving 100 mg of Ehrlich’s reagent in 5 mL of 2 N Perchloric acid. 1 mL

of Ehrlich’s reagent was combined with each of the 1 mL δ-AL-pyrrole aliquots.

Reactions with the Ehrlich’s reagent were scanned with the spectrophotometer exactly 1

min after addition of the Ehrlich’s reagent. Reactions were monitored at a ʎ = 553 nm

using a 2 mL quartz cuvette in an Agilent 8453 UV-Vis spectrophotometer over 10 min

at room temperature. The resulting Ehrlich derivative is detectable at A553, with a ϵ =

68,000 (cm M)-1.

Reactions testing δ-AL production using mALAS began with the same reaction

conditions mentioned in the succinyl-CoA regeneration system section. A 100 μL aliquot

of an overnight reaction of succinyl-CoA with glycine using mALAS was combined with

5 mL of 1 M acetic acid pH 4.6. 100 µL of acetyl acetone was added to this reaction

mixture and mixed by inversion. The reaction mixture was placed in a boiling water bath

for 10 min. Ehrlich’s reagent was prepared the same way mentioned previously. 1 mL of

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Ehrlich’s reagent was combined with 1 mL of reaction mixture and scanned with

spectrophotometer using the same conditions mentioned above.

Reactions testing δ-AL production using mALAS with a plate assay began with

the same reaction conditions mention in the succinyl-CoA regeneration system section.

200 μL of this reaction mixture was aliquoted into 8 wells of a micro titer plate and

incubated at room temperature overnight. Then a master mix containing 40 μL of acetyl

acetone and 2 mL of 1 M acetic acid pH 4.6 was prepared. 200 μL of the acetyl acetone

master mix was aliquoted into a 96 well plate. Next, 3 μL of this reaction mixture was

transferred to the 96 well plate with the acetyl acetone aliquots. The entire plate was

then submerged in a boiling water bath for 10 min. Ehrlich’s reagent was then prepared

as previously mentioned. 150 μL of Ehrlich’s reagent was aliquoted into the δ-AL-pyrrole

reaction. Reactions were scanned with a SpectraMax 190 Microplate Reader from

wavelengths between 460-780 nm.

Derivatizing Amino Acids Using PITC

Using 10 μL of a 10 mM amino acid solution was dried using a Speed Vac to

remove solvent. Dry sample pellets were dissolved in 100 μL of a coupling buffer

containing 50% acetonitrile, 25% pyridine, 10% triethylamine, 15% D.I. water. Samples

were then dried using a Speed Vac to remove solvent a second time. Dry sample

pellets were dissolved in 100 μL of the coupling buffer a second time. After

reconstitution in coupling buffer, 5 μL of PITC was added to each sample and they were

incubated at room temperature for 5 min. Samples were then dried using a Speed Vac

to remove solvent for a third time. Samples were then dissolved in 250 μL of 50 mM

NH4 Acetate pH 6.8 (solvent A for the HPLC separation).

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Detecting PITC Amino Acid Derivatives by HPLC

PITC amino acid derivatives can be separated using 150 × 4.6 mm Synergi

Hydro-RP 80 Å column using reverse phase HPLC. Solvent A was 50 mM NH4 Acetate

pH 6.8 and solvent B was 100 mM NH4 Acetate pH 6.8 with 45% acetonitrile, and 10%

methanol. Flow rate was 1 mL/min. Program began with 100 % solvent A with an

immediate increase of solvent B to 15% over 15 min, followed by an increase of solvent

B to 50% over 15 min, followed by an increase of solvent B to 100% over 4 min at which

the program stayed for 3 min, this was followed by a decrease of solvent B to 0% over 3

min at which the program remained for 10 min (HPLC method is listed as RWP2.Meth in

Appendix D). PITC amino acid derivatives eluted near 33.9 min for Phe, 31.2 min for

Leu, 6.6 min for Glu, 17.3 min for Ala, 12.0 min for Gly, and δ-AL was never determined.

Derivatizing Amino Acids Using MSTFA

Standards of δ-AL and glycine were used for derivatization with MSTFA. δ-AL

and glycine were dissolved in either D.I. water or 100 mM HEPES pH 7.5. A Speed Vac

was used to dry the samples until all solvent was removed and a pellet was formed.

Depending on the volume of the samples (typically samples were between 100-250 μL)

removing the solvent would take between 1-2 h. The pellets were resuspended with 50

μL of an 80:20 methanol / water mix. Solvent was then removed a second time using a

Speed Vac until a pellet forms again. The pellets were then resuspended again with 50

μL of methylene chloride, adding one drop at a time (usually between 5-6 drops). The

solvent was removed a third time using a Speed Vac until a pellet formed yet again.

After this succession of solvent switches was completed, 1 drop of pyridine was added

to the pellets. Next, 50 μL of MSTFA was added to the samples. Samples were placed

in a microfuge tube holder and secured into a 37°C shaker with 250 rpm of agitation for

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30 min. After samples returned to room temperature they were mixed by vortexing and

the immediately analyzed by GC-MS.

Detecting MSTFA Amino Acid Derivatives Using GC-MS

TMS-amino acid derivatives can be detected by GC-MS using a DB-17 column

(0.25 mm x 30 m). The temperature program used to detect TMS-amino acid derivatives

began with an initial temperature of 60°C for 5 min, followed by an increase at 10°C/min

to a temperature of 195°C at which the program remained for 10 min (GC method is

listed as SEF.Meth in Appendix D). TMS-glycine eluted near 9.8 min and TMS-δ-AL

eluted near 16.1 min.

Conclusions

Though we were able to make all three mALAS libraries we were not able to

screen them. We were not able to successfully overexpress and purify mALAS. Also,

we were not able to detect PITC or MSTFA derivatized δ-AL products using our reaction

conditions by GC or HPLC. These are both areas which require further optimization.

Should we never successfully optimize the MSTFA assay to work with our reaction

conditions, or devise a new method for directly detecting our products, we do have other

options. We were able to get several analytical methods to work using the mALAS

reaction conditions during this project. One such method was the spectrophotometric

assay using the α-KGD regeneration system. This assay is the most reliable method we

have for detecting mALAS activity for amino acid substitutes for glycine. So if the

MSTFA protocol cannot be successfully optimized then this spectrophotometric assay

would be our best method for detecting products when we use substitutes for glycine.

However, though we can substitute for glycine with this assay, we cannot use it with any

substrate other than succinyl-CoA, and we would like to substitute different acyl-CoA

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substrates for succinyl-CoA. So should it become necessary, we can separate acyl-CoA

substrates from CoA by HPLC. Using this method we could obtain the ratios of

substrates to products. Of course screening mutants with either method would require

us to successfully purify the mALAS mutants. And as such, it would be the most

immediate protocol we would need to optimize.

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Table 5-1. Retention times of PIT-amino acid derivative standards from HPLC.

Amino Acid Standard HPLC retention times of PIT-amino acid standards from assay (min)

HPLC retention times PIT-amino acid standards from the literature (min)

PIT-Phenylalanine 33.1 32.0

PIT-Alanine 17.3 15.5

PIT-Glycine 12.0 10.0

PIT-Glutamate 6.6 4.5

PIT-Leucine 31.2 30.0

PIT-δ-AL Not detected Not reported

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Figure 5-1. The reaction of glycine and succinyl-CoA to make δ-AL using ALAS as a

catalyst.

211

Figure 5-2. The proposed mechanism of ALAS.

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Figure 5-3. The active site of ALAS from R. capsulatus with glycine bound to PLP (2BWP). The hydrophobic positions of interest for protein engineering are shown in orange and the values in parenthesis are the positions in the mALAS structure. The glycine loop is shown in blue and glycine bound to PLP is shown in yellow.

Figure 5-4. The active site of ALAS from R. capsulatus with succinyl-CoA (2BWO). The arginine position of interest for protein engineering is shown in orange.

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Figure 5-5. Reaction scheme of the derivatizing of δ-AL-pyrrole with Ehrlich’s reagent.

Figure 5-6. Reaction scheme of succinic anhydride with CoA to make succinyl-CoA.

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Figure 5-7. The coupling of ALAS production of CoA from succinyl-CoA to α-Ketoglutarate Dehydrogenase production of NADH from NAD+.

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Figure 5-8. Derivatization of amino acids using PITC.

Figure 5-9. Derivatization of amino acids using MSTFA.

Figure 5-10. The results for the reaction of δ-AL-pyrrole with Ehrlich’s reagent. These results were obtained from reactions using mALAS. The reaction that ran overnight using mALAS gave three times the product as the reaction that was run for 1 h. This chart also shows that it takes only about 5 mins for the reaction of δ-AL-pyrrole with Ehrlich’s reagent to go to completion.

216

Figure 5-11. The results for the reaction of δ-AL-pyrrole with Ehrlich’s reagent using a plate reader. Eight redundant reactions were run overnight with mALAS wt. using the regeneration system. Reactions were scanned with a SpectraMax 190 Microplate Reader from wavelengths between 460-780 nm. The Ehrlich’s derivative absorbs very strongly near 553 nm. Our sample with δ-AL-pyrrole is slightly blue shifted. All samples gave identical results, this figure shows the results for one sample.

217

Figure 5-12. The reaction of succinyl-CoA with hydroxyl amine to displace the CoA.

Figure 5-13. HPLC results from the reaction of succinic anhydride and CoA to make succinyl-CoA. Succinyl-CoA Rt = 15.5 mins, CoA Rt = 14.9 mins, & caffeine Rt = 24.3 mins. This chromatograph shows our reaction to make Succinyl-CoA has gone to completion.

218

Figure 5-14. HPLC results from the synthesis of succinyl-CoA from succinic anhydride and CoA co-eluted with 10x CoA. Succinyl-CoA Rt = 15.5 mins, CoA Rt = 14.9 mins, & caffeine Rt = 24.3 mins. This chromatograph shows our completed reaction spiked with 10x CoA standard and verifies the location of CoA.

219

Figure 5-15. HPLC results from the reaction of succinyl-CoA with hydroxylamine. Succinyl-CoA Rt = 15.5 mins, & CoA Rt = 14.9 mins. Using an aliquot of our completed succinyl-CoA reaction, we ran the hydroxyl amine to displace the CoA. What this chromatograph shows is that the succinyl-CoA has been completely displaced and the CoA peak reappears.

220

Figure 5-16. Results of the succinyl-CoA regeneration system. Α-KGD takes less than a minute to convert all the CoA into succinyl-CoA. At this point the NADH concentration detected is equivalent to the maximum concentration of CoA (NADH maximum which is the orange line). Hydrolysis of succinyl-CoA back to CoA in solution is what causes the slow increase afterwards.

221

Figure 5-17. Results of reactions from the succinyl-CoA regeneration system using mALAS and mALAS R433K. Both mALAS Wt and the mALAS R433K batches covert glycine to δ-AL and that detection is indirectly monitored using the α-KGD regeneration system.

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Figure 5-18. Results of amino acid derivatization with MSTFA. These results are from the standards of mixtures of glycine and δ-AL. Neither enzyme, buffer, nor any other component was present in these standards.

223

APPENDIX A LIST OF PRIMERS

Chapter 1 Primers

Table A-1. List of mutagenic primers for Chapter 1.

Mutation Sequence

Y196X Fwd 5’-GTG CTA ACG GTN NKT TGT TAA ACC AGT TCT TGG ACC CTC A-3’

Y196X Rev 5’-TGG TTT AAC AAM NNA CCG TTA GCA CTG TGA ATT TCA ACA C-3’

Y196W Fwd 5’-GTG CTA ACG GTT GGT TGT TAA ACC AGT TCT TGG ACC CTC A-3’

Y196W Rev 5’-TGG TTT AAC AAC CAA CCG TTA GCA CTG TGA ATT TCA ACA C-3’

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Chapter 2 Primers

Table A-2. List of mutagenic and sequencing primers for Chapter 2.

Mutation Sequence

Y78T Fwd 5’-AAG CCT CTG GTA CTG AAG GTG CTG CTC CAG GTA TTT GGA C-3’

Y78T Rev 5’-GCA GCA CCT TCA GTA CCA GAG GCT TGA GGA GAG ACA AAA G-3’

Y78V Fwd 5’-AAG CCT CTG GTG TTG AAG GTG CTG CTC CAG GTA TTT GGA C-3’

Y78V Rev 5’-GCA GCA CCT TCA ACA CCA GAG GCT TGA GGA GAG ACA AAA G-3’

Y78XKST Fwd 5’-AAG CCT CTG GTK STG AAG GTG CTG CTC CAG GTA TTT GGA C-3’

Y78XKST Rev 5’-GCA GCA CCT TCA TAA CCA GAG GCT TGA GGA GAG ACA AAA G-3’

I113A Fwd 5’-CAA CCC AGT TGG CTT TTT TGG GAA GGG TTG CAG ATC CAG C-3’

I113A Rev 5’-CTT CCC AAA AAA GCC AAC TGG GTT GAA ACG AAA GAA CCG T-3’

I113C Fwd 5’-CAA CCC AGT TGT GTT TTT TGG GAA GGG TTG CAG ATC CAG C-3’

I113C Rev 5’-CTT CCC AAA AAA CAC AAC TGG GTT GAA ACG AAA GAA CCG T-3’

I113G Fwd 5’-CAA CCC AGT TGG GTT TTT TGG GAA GGG TTG CAG ATC CAG C-3’

I113G Rev 5’-CTT CCC AAA AAA CCC AAC TGG GTT GAA ACG AAA GAA CCG T-3’

I113S Fwd 5’-CAA CCC AGT TGT CTT TTT TGG GAA GGG TTG CAG ATC CAG C-3’

I113S Rev 5’-CTT CCC AAA AAA GAC AAC TGG GTT GAA ACG AAA GAA CCG T-3’

I113T Fwd 5’-CAA CCC AGT TGA CTT TTT TGG GAA GGG TTG CAG ATC CAG C-3’

I113T Rev 5’-CTT CCC AAA AAA GTC AAC TGG GTT GAA ACG AAA GAA CCG T-3’

I113V Fwd 5’-CAA CCC AGT TGG TTT TTT TGG GAA GGG TTG CAG ATC CAG C-3’

I113V Rev 5’-CTT CCC AAA AAA ACC AAC TGG GTT GAA ACG AAA GAA CCG T-3’

I113XKST Fwd 5’-CAA CCC AGT TGK STT TTT TGG GAA GGG TTG CAG ATC CAG C-3’

I113XKST Rev 5’-CTT CCC AAA AAA SMC AAC TGG GTT GAA ACG AAA GAA CCG T-3’

F247X Fwd 5’-CAT GGG CTA CTN NKC AAA ACA TGA AGG CTC ACA AGG ACA C-3’

F247X Rev 5’-TTC ATG TTT TGM NNA GTA GCC CAT GGA GAG ATT CTG ATA C-3’

OYE 2.6 Seq 5’-TCC AGC AAG TAT ATA GCA TGG CCT-3’

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Chapter 3 Primers


Mutation Sequence

E41X Fwd 5’-TTA GAG CTT TAN NKG ACC ACA CTC CTT CTG ATT TGC AAT T-3’

E41X Rev 5’-GGA GTG TGG TCM NNT AAA GCT CTA AAT CTA GTA GTT GGT G-3’

D141X Fwd 5’-CTT ATG AAA GTN NKG CCG CTA AAG AAG CTG CCG AAG CAG T-3’

D141X Rev 5’-TCT TTA GCG GCM NNA CTT TCA TAA GTA GCA GAG GCA GAA A-3’

E145X Fwd 5’-ATG CCG CTA AAN NKG CTG CCG AAG CAG TTG GTA ACC CTG T-3’

E145X Rev 5’-GCT TCG GCA GCM NNT TTA GCG GCA TCA CTT TCA TAA GTA G-3’

K330X Fwd 5’- CTC CAG AGT TCN NKA CAT TGA AGG AAG ATA TCG CTG ACA A-3’

K330X Rev 5’-TCC TTC AAT GTM NNG AAC TCT GGA GCA TCG TAG GAG TAG T-3’

I214X Fwd 5’-ACG GTG GAT CCN NKG AGA ACA GAG CCA GGT TAA TTC TTG A-3’

I214X Rev 5’-GCT CTG TTC TCM NNG GAT CCA CCG TAT TCA TCA GTT CTT T-3’

W244X Fwd 5’-GAA TCT CTC CAN NKG CTA CTT TCC AAA ACA TGA AGG CTC A-3’

W244X Rev 5’-TGG AAA GTA GCM NNT GGA GAG ATT CTG ATA CCA ATC TTG T-3’

L260X Fwd 5’- CTG TTC ACC CAN NKA CTA CTT TCT CTT ACT TGG TCC ACG A -3’

L260X Rev 5’-GAG AAA GTA GTM NNT GGG TGA ACA GTG TCC TTG TGA GCC T-3’

F307X Fwd 5’-GTG ACA ACG AAN NKG TCT CCA AGA TCT GGA AGG GTG TTA T-3’

F307X Rev 5’-ATC TTG GAG ACM NNT TCG TTG TCA CCA GCT TGG TCT TCT T-3’

I311X Fwd 5’-TTG TCT CCA AGN NKT GGA AGG GTG TTA TCT TGA AGG CAG G-3’

I311X Rev 5’-ACA CCC TTC CAM NNC TTG GAG ACA AAT TCG TTG TCA CCA G-3’

S388P Fwd 5’-TTT CTA TGG ATC CGG AAG AGG TTG ATA AAG AAT TAG AAA T-3’

S388P Rev 5’-TCA ACC TCT TCC GGA TCC ATA GAA AAG GTA TTG TAA CCA G-3’

S388I Fwd 5’-TTT CTA TGG ATA TTG AAG AGG TTG ATA AAG AAT TAG AAA T-3’

S388I Rev 5’-TCA ACC TCT TCA ATA TCC ATA GAA AAG GTA TTG TAA CCA T-3’

S388N Fwd 5’-TTT CTA TGG ATA ATG AAG AGG TTG ATA AAG AAT TAG AAA T-3’

S388N Rev 5’-TCA ACC TCT TCA TTA TCC ATA GAA AAG GTA TTG TAA CCA T-3’

S388Y Fwd 5’-TTT CTA TGG ATT ATG AAG AGG TTG ATA AAG AAT TAG AAA T-3’

S388Y Rev 5’-TCA ACC TCT TCA TAA TCC ATA GAA AAG GTA TTG TAA CCA T-3’

OYE 2.6 Seq 5’-TCC AGC AAG TAT ATA GCA TGG CCT-3’’

226

Chapter 4 Primers


Mutation Sequence

Y389Y Fwd 5’-ACT ACC CAA CAT ACG AAG AGG CAG TAG ATT TAG GTT GGA A-3’

Y389Y Rev 5’-ACT GCC TCT TCG TAT GTT GGG TAG TCG GTA TAA CCT TCC G-3’

NdeI(-) Fwd 5’-AAG GAG ATA TAC AAA TGC CCA TGG ATA TCG GAA TTA ATT C-3’

NdeI(-) Rev 5’-TCC ATG GGC ATT TGT ATA TCT CCT TCT TAA AGT TAA ACA A-3’

NdeI(+)Fwd 5’-ATC CGA ATT CGC ATA TGC CAT TTG TAA AAG GTT TTG AGC C-3’

NdeI(+)Rev 5’-ACA AAT GGC ATA TGC GAA TTC GGA TCC GAA TTA ATT CCG A-3’

F296S Fwd 5’-GTA ACT AAC CCA TCC TTG ACT GAA GGG GAG GGT GAA TAC G-3’

F296S Rev 5’-CCT TCA GTC AAG GAT GGG TTA GTT ACA CGA GGT TCA ACC A-3’

W116A Fwd 5’-GGG TAC AAC TTG CTT CTT TAG GCT GGG CAT CCT TCC CAG A-3’

W116A Rev 5’-CAG CCT AAA GAA GCA AGT TGT ACC CAC GCG AAC GAC TGA C-3’

W116C Fwd 5’-GGG TAC AAC TTT GTT CTT TAG GCT GGG CAT CCT TCC CAG A-3’

W116C Rev 5’-CAG CCT AAA GAA CAA AGT TGT ACC CAC GCG AAC GAC TGA C-3’

W116D Fwd 5’-GGG TAC AAC TTG ATT CTT TAG GCT GGG CAT CCT TCC CAG A-3’

W116D Rev 5’-CAG CCT AAA GAA TCA AGT TGT ACC CAC GCG AAC GAC TGA C-3’

W116E Fwd 5’-GGG TAC AAC TTG AAT CTT TAG GCT GGG CAT CCT TCC CAG A-3’

W116E Rev 5’-CAG CCT AAA GAT TCA AGT TGT ACC CAC GCG AAC GAC TGA C-3’

W116F Fwd 5’-GGG TAC AAC TTT TTT CTT TAG GCT GGG CAT CCT TCC CAG A-3’

W116F Rev 5’-CAG CCT AAA GAC CAA AGT TGT ACC CAC GCG AAC GAC TGA C-3’

W116G Fwd 5’-GGG TAC AAC TTG GGT CTT TAG GCT GGG CAT CCT TCC CAG A-3’

W116G Rev 5’-CAG CCT AAA GAA CGA AGT TGT ACC CAC GCG AAC GAC TGA C-3’

W116H Fwd 5’-GGG TAC AAC TTC ATT CTT TAG GCT GGG CAT CCT TCC CAG A-3’

W116H Rev 5’-CAG CCT AAA GAA TGA AGT TGT ACC CAC GCG AAC GAC TGA C-3’

W116I Fwd 5’-GGG TAC AAC TTA TTT CTT TAG GCT GGG CAT CCT TCC CAG A-3’

W116I Rev 5’-CAG CCT AAA GAA ATA AGT TGT ACC CAC GCG AAC GAC TGA C-3’

W116K Fwd 5’-GGG TAC AAC TTA AAT CTT TAG GCT GGG CAT CCT TCC CAG A-3’

W116K Rev 5’-CAG CCT AAA GAT TTA AGT TGT ACC CAC GCG AAC GAC TGA C-3’

W116L Fwd 5’-GGG TAC AAC TTC TTT CTT TAG GCT GGG CAT CCT TCC CAG A-3’

W116L Rev 5’-CAG CCT AAA GAA AGA AGT TGT ACC CAC GCG AAC GAC TGA C-3’

W116M Fwd 5’-GGG TAC AAC TTA TGT CTT TAG GCT GGG CAT CCT TCC CAG A-3’

W116M Rev 5’-CAG CCT AAA GAC ATA AGT TGT ACC CAC GCG AAC GAC TGA C-3’

W116N Fwd 5’-GGG TAC AAC TTA ATT CTT TAG GCT GGG CAT CCT TCC CAG A-3’

W116N Rev 5’-CAG CCT AAA GAA TTA AGT TGT ACC CAC GCG AAC GAC TGA C-3’

W116P Fwd 5’-GGG TAC AAC TTC CTT CTT TAG GCT GGG CAT CCT TCC CAG A-3’

227

Table A-4. Continued

Mutation Sequence

W116P Rev 5’-CAG CCT AAA GAA GGA AGT TGT ACC CAC GCG AAC GAC TGA C-3’

W116Q Fwd 5’-GGG TAC AAC TTC AAT CTT TAG GCT GGG CAT CCT TCC CAG A-3’

W116Q Rev 5’-CAG CCT AAA GAT TGA AGT TGT ACC CAC GCG AAC GAC TGA C-3’

W116R Fwd 5’-GGG TAC AAC TTC GTT CTT TAG GCT GGG CAT CCT TCC CAG A-3’

W116R Rev 5’-CAG CCT AAA GAA CGA AGT TGT ACC CAC GCG AAC GAC TGA C-3’

W116S Fwd 5’-GGG TAC AAC TTT CTT CTT TAG GCT GGG CAT CCT TCC CAG A-3’

W116S Rev 5’-CAG CCT AAA GAA GAA AGT TGT ACC CAC GCG AAC GAC TGA C-3’

W116T Fwd 5’-GGG TAC AAC TTA CTT CTT TAG GCT GGG CAT CCT TCC CAG A-3’

W116T Rev 5’-CAG CCT AAA GAA GTA AGT TGT ACC CAC GCG AAC GAC TGA C-3’

W116V Fwd 5’-GGG TAC AAC TTG TTT CTT TAG GCT GGG CAT CCT TCC CAG A-3’

W116V Rev 5’-CAG CCT AAA GAA ACA AGT TGT ACC CAC GCG AAC GAC TGA C-3’

W116Y Fwd 5’-GGG TAC AAC TTT ATT CTT TAG GCT GGG CAT CCT TCC CAG A-3’

W116Y Rev 5’-CAG CCT AAA GAA TAA AGT TGT ACC CAC GCG AAC GAC TGA C-3’

OYE 3 seq 5’-AGC GTT TGG CCT TTG TGC ACC TCG-3’

228

Chapter 5 Primers


Mutation Sequence

R85A Fwd 5’-ACC ACA CCT ACG CTG TGT TCA AGA CTG TGA ATC GTT GGG C-3’

R85A Rev 5’-GTC TTG AAC ACA GCG TAG GTG TGG TCC TGT TTC TTC TCC A-3’

R85C Fwd 5’-ACC ACA CCT ACT GTG TGT TCA AGA CTG TGA ATC GTT GGG C-3’

R85C Rev 5’-GTC TTG AAC ACA CAG TAG GTG TGG TCC TGT TTC TTC TCC A-3’

R85D Fwd 5’-ACC ACA CCT ACG ATG TGT TCA AGA CTG TGA ATC GTT GGG C-3’

R85D Rev 5’-GTC TTG AAC ACA TCG TAG GTG TGG TCC TGT TTC TTC TCC A-3’

R85E Fwd 5’-ACC ACA CCT ACG AAG TGT TCA AGA CTG TGA ATC GTT GGG C-3’

R85E Rev 5’-GTC TTG AAC ACT TCG TAG GTG TGG TCC TGT TTC TTC TCC A-3’

R85F Fwd 5’- ACC ACA CCT ACT TTG TGT TCA AGA CTG TGA ATC GTT GGG C-3

R85F Rev 5’- GTC TTG AAC ACA AAG TAG GTG TGG TCC TGT TTC TTC TCC A-3

R85G Fwd 5’- ACC ACA CCT ACG GGG TGT TCA AGA CTG TGA ATC GTT GGG C-3

R85G Rev 5’-GTC TTG AAC ACC CCG TAG GTG TGG TCC TGT TTC TTC TCC A-3’

R85H Fwd 5’- ACC ACA CCT ACC ATG TGT TCA AGA CTG TGA ATC GTT GGG C-3’

R85H Rev 5’- GTC TTG AAC ACA TGG TAG GTG TGG TCC TGT TTC TTC TCC A-3’

R85I Fwd 5’-ACC ACA CCT ACA TTG TGT TCA AGA CTG TGA ATC GTT GGG C-3’

R85I Rev 5’-GTC TTG AAC ACA ATG TAG GTG TGG TCC TGT TTC TTC TCC A-3’

R85K Fwd 5’-ACC ACA CCT ACA AAG TGT TCA AGA CTG TGA ATC GTT GGG C-3’

R85K Rev 5’-GTC TTG AAC ACT TTG TAG GTG TGG TCC TGT TTC TTC TCC A-3’

R85L Fwd 5’-ACC ACA CCT ACC TTG TGT TCA AGA CTG TGA ATC GTT GGG C-3’

R85L Rev 5’-GTC TTG AAC ACA AGG TAG GTG TGG TCC TGT TTC TTC TCC A-3’

R85M Fwd 5’-ACC ACA CCT ACA TGG TGT TCA AGA CTG TGA ATC GTT GGG C-3’

R85M Rev 5’-GTC TTG AAC ACC ATG TAG GTG TGG TCC TGT TTC TTC TCC A-3’

R85N Fwd 5’-ACC ACA CCT ACA ATG TGT TCA AGA CTG TGA ATC GTT GGG C-3’

R85N Rev 5’-GTC TTG AAC ACA TTG TAG GTG TGG TCC TGT TTC TTC TCC A-3’

R85P Fwd 5’-ACC ACA CCT ACC CTG TGT TCA AGA CTG TGA ATC GTT GGG C-3’

R85P Rev 5’-GTC TTG AAC ACA GGG TAG GTG TGG TCC TGT TTC TTC TCC A-3’

R85Q Fwd 5’-ACC ACA CCT ACC AAG TGT TCA AGA CTG TGA ATC GTT GGG C-3’

R85Q Rev 5’-GTC TTG AAC ACT TGG TAG GTG TGG TCC TGT TTC TTC TCC A-3’

R85S Fwd 5’-ACC ACA CCT ACA GTG TGT TCA AGA CTG TGA ATC GTT GGG C-3’

R85S Rev 5’-GTC TTG AAC ACA CTG TAG GTG TGG TCC TGT TTC TTC TCC A-3’

R85T Fwd 5’-ACC ACA CCT ACA CTG TGT TCA AGA CTG TGA ATC GTT GGG C-3’

R85T Rev 5’-GTC TTG AAC ACA GTG TAG GTG TGG TCC TGT TTC TTC TCC A-3’

R85V Fwd 5’-ACC ACA CCT ACG TTG TGT TCA AGA CTG TGA ATC GTT GGG C-3’

229


Mutation Sequence

R85V Rev 5’-GTC TTG AAC ACA ACG TAG GTG TGG TCC TGT TTC TTC TCC A-3’

R85W Fwd 5’-ACC ACA CCT ACT GGG TGT TCA AGA CTG TGA ATC GTT GGG C-3’

R85W Rev 5’-GTC TTG AAC ACC CAG TAG GTG TGG TCC TGT TTC TTC TCC A-3’

R85Y Fwd 5’-ACC ACA CCT ACT ATG TGT TCA AGA CTG TGA ATC GTT GGG C-3’

R85Y Rev 5’-GTC TTG AAC ACA TAG TAG GTG TGG TCC TGT TTC TTC TCC A-3’

T148A Fwd 5’-GAG CTG GGG GCG CTC GCA ATA TCT CAG GTA CCA GCA AGT T-3’

T148A Rev 5’-GAG ATA TTG CGA GCG CCC CCA GCT CCA GCT CCA TGA TTC T-3’

T148C Fwd 5’-GAG CTG GGG GCT GTC GCA ATA TCT CAG GTA CCA GCA AGT T-3’

T148C Rev 5’-GAG ATA TTG CGA CAG CCC CCA GCT CCA GCT CCA TGA TTC T-3’

T148D Fwd 5’-GAG CTG GGG GCG ATC GCA ATA TCT CAG GTA CCA GCA AGT T-3’

T148D Rev 5’-GAG ATA TTG CGA TCG CCC CCA GCT CCA GCT CCA TGA TTC T-3’

T148E Fwd 5’-GAG CTG GGG GCG AGC GCA ATA TCT CAG GTA CCA GCA AGT T-3’

T148E Rev 5’-GAG ATA TTG CGC TCG CCC CCA GCT CCA GCT CCA TGA TTC T-3’

T148F Fwd 5’-GAG CTG GGG GCT TTC GCA ATA TCT CAG GTA CCA GCA AGT T-3’

T148F Rev 5’-GAG ATA TTG CGA AAG CCC CCA GCT CCA GCT CCA TGA TTC T-3’

T148G Fwd 5’-GAG CTG GGG GCG GTC GCA ATA TCT CAG GTA CCA GCA AGT T-3’

T148G Rev 5’-GAG ATA TTG CGA CCG CCC CCA GCT CCA GCT CCA TGA TTC T-3’

T148H Fwd 5’-GAG CTG GGG GCC ATC GCA ATA TCT CAG GTA CCA GCA AGT T-3’

T148H Rev 5’-GAG ATA TTG CGA TGG CCC CCA GCT CCA GCT CCA TGA TTC T-3’

T148I Fwd 5’-GAG CTG GGG GCA TTC GCA ATA TCT CAG GTA CCA GCA AGT T-3’

T148I Rev 5’-GAG ATA TTG CGA ATG CCC CCA GCT CCA GCT CCA TGA TTC T-3’

T148K Fwd 5’-GAG CTG GGG GCA AAC GCA ATA TCT CAG GTA CCA GCA AGT T-3’

T148K Rev 5’-GAG ATA TTG CGT TTG CCC CCA GCT CCA GCT CCA TGA TTC T-3’

T148L Fwd 5’-GAG CTG GGG GCC TTC GCA ATA TCT CAG GTA CCA GCA AGT T-3’

T148L Rev 5’-GAG ATA TTG CGA AGG CCC CCA GCT CCA GCT CCA TGA TTC T-3’

T148M Fwd 5’-GAG CTG GGG GCA TGC GCA ATA TCT CAG GTA CCA GCA AGT T-3’

T148M Rev 5’-GAG ATA TTG CGC ATG CCC CCA GCT CCA GCT CCA TGA TTC T-3’

T148N Fwd 5’-GAG CTG GGG GCA ATC GCA ATA TCT CAG GTA CCA GCA AGT T-3’

T148N Rev 5’-GAG ATA TTG CGA TTG CCC CCA GCT CCA GCT CCA TGA TTC T-3’

T148P Fwd 5’-GAG CTG GGG GCC CTC GCA ATA TCT CAG GTA CCA GCA AGT T-3’

T148P Rev 5’-GAG ATA TTG CGA GGG CCC CCA GCT CCA GCT CCA TGA TTC T-3’

T148Q Fwd 5’-GAG CTG GGG GCC AAC GCA ATA TCT CAG GTA CCA GCA AGT T-3’

T148Q Rev 5’-GAG ATA TTG CGT TGG CCC CCA GCT CCA GCT CCA TGA TTC T-3’

T148R Fwd 5’-GAG CTG GGG GCA GAC GCA ATA TCT CAG GTA CCA GCA AGT T-3’

T148R Rev 5’-GAG ATA TTG CGT CTG CCC CCA GCT CCA GCT CCA TGA TTC T-3’

230


Mutation Sequence

T148S Fwd 5’-GAG CTG GGG GCA GTC GCA ATA TCT CAG GTA CCA GCA AGT T-3’

T148S Rev 5’-GAG ATA TTG CGA CTG CCC CCA GCT CCA GCT CCA TGA TTC T-3’

T148V Fwd 5’-GAG CTG GGG GCG TTC GCA ATA TCT CAG GTA CCA GCA AGT T-3’

T148V Rev 5’-GAG ATA TTG CGA AGG CCC CCA GCT CCA GCT CCA TGA TTC T-3’

T148W Fwd 5’-GAG CTG GGG GCT GGC GCA ATA TCT CAG GTA CCA GCA AGT T-3’

T148W Rev 5’-GAG ATA TTG CGC CAG CCC CCA GCT CCA GCT CCA TGA TTC T-3’

T148Y Fwd 5’-GAG CTG GGG GCT ATC GCA ATA TCT CAG GTA CCA GCA AGT T-3’

T148Y Rev 5’-GAG ATA TTG CGA TAG CCC CCA GCT CCA GCT CCA TGA TTC T-3’

I151A Fwd 5’-GCA CTC GCA ATG CCT CAG GTA CCA GCA AGT TTC ATG TGG A-3’

I151A Rev 5’-CTG GTA CCT GAG GCA TTG CGA GTG CCC CCA GCT CCA GCT C-3’

I151C Fwd 5’-GCA CTC GCA ATT GCT CAG GTA CCA GCA AGT TTC ATG TGG A-3’

I151C Rev 5’-CTG GTA CCT GAG CAA TTG CGA GTG CCC CCA GCT CCA GCT C-3’

I151D Fwd 5’-GCA CTC GCA ATG ACT CAG GTA CCA GCA AGT TTC ATG TGG A-3’

I151D Rev 5’-CTG GTA CCT GAG TCA TTG CGA GTG CCC CCA GCT CCA GCT C-3’

I151E Fwd 5’-GCA CTC GCA ATG AGT CAG GTA CCA GCA AGT TTC ATG TGG A-3’

I151E Rev 5’-CTG GTA CCT GAC TCA TTG CGA GTG CCC CCA GCT CCA GCT C-3’

I151F Fwd 5’-GCA CTC GCA ATT TCT CAG GTA CCA GCA AGT TTC ATG TGG A-3’

I151F Rev 5’-CTG GTA CCT GAG AAA TTG CGA GTG CCC CCA GCT CCA GCT C-3’

I151G Fwd 5’-GCA CTC GCA ATG GCT CAG GTA CCA GCA AGT TTC ATG TGG A-3’

I151G Rev 5’-CTG GTA CCT GAG CCA TTG CGA GTG CCC CCA GCT CCA GCT C-3’

I151H Fwd 5’-GCA CTC GCA ATC ACT CAG GTA CCA GCA AGT TTC ATG TGG A-3’

I151H Rev 5’-CTG GTA CCT GAG TGA TTG CGA GTG CCC CCA GCT CCA GCT C-3’

I151K Fwd 5’-GCA CTC GCA ATA AGT CAG GTA CCA GCA AGT TTC ATG TGG A-3’

I151K Rev 5’-CTG GTA CCT GAC TTA TTG CGA GTG CCC CCA GCT CCA GCT C-3’

I151M Fwd 5’-GCA CTC GCA ATA TGT CAG GTA CCA GCA AGT TTC ATG TGG A-3’

I151M Rev 5’-CTG GTA CCT GAC ATA TTG CGA GTG CCC CCA GCT CCA GCT C-3’

I151N Fwd 5’-GCA CTC GCA ATA ACT CAG GTA CCA GCA AGT TTC ATG TGG A-3’

I151N Rev 5’-CTG GTA CCT GAG TTA TTG CGA GTG CCC CCA GCT CCA GCT C-3’

I151P Fwd 5’-GCA CTC GCA ATC CCT CAG GTA CCA GCA AGT TTC ATG TGG A-3’

I151P Rev 5’-CTG GTA CCT GAG GGA TTG CGA GTG CCC CCA GCT CCA GCT C-3’

I151Q Fwd 5’- GCA CTC GCA ATA ACT CAG GTA CCA GCA AGT TTC ATG TGG A-3’

I151Q Rev 5’- CTG GTA CCT GAG TTA TTG CGA GTG CCC CCA GCT CCA GCT C-3’

I151R Fwd 5’- GCA CTC GCA ATA GGT CAG GTA CCA GCA AGT TTC ATG TGG A-3’

I151R Rev 5’-CTG GTA CCT GAC CTA TTG CGA GTG CCC CCA GCT CCA GCT C-3’

I151S Fwd 5’-GCA CTC GCA ATA GCT CAG GTA CCA GCA AGT TTC ATG TGG A-3’

231


Mutation Sequence

I151S Rev 5’-CTG GTA CCT GAG CTA TTG CGA GTG CCC CCA GCT CCA GCT C-3’

I151T Fwd 5’-GCA CTC GCA ATA CCT CAG GTA CCA GCA AGT TTC ATG TGG A-3’

I151T Rev 5’-CTG GTA CCT GAG GTA TTG CGA GTG CCC CCA GCT CCA GCTC-3’

I151V Fwd 5’-GCA CTC GCA ATG TCT CAG GTA CCA GCA AGT TTC ATG TGG A-3’

I151V Rev 5’-CTG GTA CCT GAG ACA TTG CGA GTG CCC CCA GCT CCA GCT C-3’

I151W Fwd 5’-GCA CTC GCA ATT GGT CAG GTA CCA GCA AGT TTC ATG TGG A-3’

I151W Rev 5’-CTG GTA CCT GAC CAA TTG CGA GTG CCC CCA GCT CCA GCT C-3’

I151Y Fwd 5’-GCA CTC GCA ATT ACT CAG GTA CCA GCA AGT TTC ATG TGG A-3’

I151Y Rev 5’-CTG GTA CCT GAG TAA TTG CGA GTG CCC CCA GCT CCA GCT C-3’

L161L Fwd 5’-GGG TAC AAC TTT ATT CTT TAG GCT GGG CAT CCT TCC CAG A-3’

L161L Rev 5’-CAG CCT AAA GAA TAA AGT TGT ACC CAC GCG AAC GAC TGA C-3’

232

APPENDIX B

MUTAGENIC PLASMIDS

Table B-1. Mutagenic plasmids used in this study.

Plasmid Parent Mutation Description

pET3b T-7 promoter, and AmpR

pET3b-OYE pET3b OYE 1 gene OYE 1 non-tagged protein

pET30a T-7 promoter, His-Tag, and KanR

pET30a-OYE pET30a OYE 1 gene OYE 1 His-tag protein

pET21a(+) T-7 promoter, and AmpR

pDJB32 pET21a(+) OYE 2.6-GST fusion protein

OYE 2.6-GST fusion protein

pBS2 pDJB32 Y368 silent Deletion of NdeI restriction site

pET22b(+) T-7 promoter, and AmpR

pFBI pET22b(+) OYE 2.6 gene OYE 2.6 non-tagged protein

pYEX-41

pET26(+) T-7 promoter, and KanR

pDJB6 pET26(+)

pGEX

OYE 3-GST fusion protein OYE 3-GST fusion protein

pRP1 pDJB6 Y389 silent Deletion of NdeI restriction site

pRP2 pRP1 5’-CAAATG-3’ Deletion of NdeI restriction site

pRP3 pRP2 5’-CATATG-3’ Addition of NdeI restriction site

pET22b(+) T-7 promoter, and AmpR

pRP4 pET22(+) OYE 3 gene OYE 3 non-tagged protein

CASS-3 phoA promoter, and AmpR

pGF23 CASS-3 mALAS gene mALAS-GST fusion protein

pRP5 pGF23 L161 silent Deletion of KpnI restriction site

233

APPENDIX C PLASMID SEQUENCES

Sequence of pET3b-OYE1

1 TTCTCATGTT TGACAGCTTA TCATCGATAA GCTTTAATGC GGTAGTTTAT

51 CACAGTTAAA TTGCTAACGC AGTCAGGCAC CGTGTATGAA ATCTAACAAT

101 GCGCTCATCG TCATCCTCGG CACCGTCACC CTGGATGCTG TAGGCATAGG

151 CTTGGTTATG CCGGTACTGC CGGGCCTCTT GCGGGATATC GTCCATTCCG

201 ACAGCATCGC CAGTCACTAT GGCGTGCTGC TAGCGCTATA TGCGTTGATG

251 CAATTTCTAT GCGCACCCGT TCTCGGAGCA CTGTCCGACC GCTTTGGCCG

301 CCGCCCAGTC CTGCTCGCTT CGCTACTTGG AGCCACTATC GACTACGCGA

351 TCATGGCGAC CACACCCGTC CTGTGGATAT CCGGATATAG TTCCTCCTTT

401 CAGCAAAAAA CCCCTCAAGA CCCGTTTAGA GGCCCCAAGG GGTTATGCTA

451 GTTATTGCTC AGCGGTGGCA GCAGCCAACT CAGCTTCCTT TCGGGCTTTG

501 TTAGCAGCCG GATCCCGACC CATTTGCTGT CCACCAGTCA TGCTAGCCAT

551 ATGCCTTTGT TAGATCAGGA ACGCCATATC TGGCAAATAT GAATACTGCT

601 CGAAGTGGTA AAAACATTCA AAGTACGCTC TCTTAAAAGT GATACAAAGG

651 TGAGCTATGA AACTGGCATT GCTTCAACGT GATAGCTGAG CCCATATTCT

701 TTCATATATG ATTTTCACGT CTGATTTTTT AATAAAAGAT GCAGAGTGAA

751 AACATGCTCC TGCCCTACTG TGTGCATATT CAGCGCTCGT CAAAAAACCT

801 GTTGATATTT ATTTTCATTA AAATTGTTTA TCCTAAATAT ATCTAGTCGT

851 TAATTATATA AGGTAAACGG GATTGTGGGG GTGGGGAAGG TAGAACATCG

901 AACTATGGAA TATTCAATTA CTTTTTGTCC CAGCCTAATT TGAGAGCTTC

951 TTCATAGGTG GGGTAGTCAA TATAACCATG AGCAGACATC TGGTAGAAAG

1001 TATCTCTGTC ATATTTGTTC AGAGGTAGAC CTTTTTCCAA ACGATCAACC

1051 AAATCCGGGT TAGAAATGAA GAATCTACCG TAACCGATCA AGGTTCTCTT

1101 GTCCTTAACT TCTTCTCTAA CGACTTCTGG GTGGAGAGCA AAATTACCAG

1151 CTCTAATGAC TGGGCCCTTC CAGATGGAGT AAACAAAATC GTTGCTACCT

1201 CCTTCGTATT CACCCTCCCC TTCAGTCAAG AATGGGTTAG TTACACGAGG

1251 TTCAACCAAA TGAACAAAAG CTAAACGTTT TCCGGCTTTA GCTCTCTTTT

1301 CTAATTCACC AGCAACGTAA GCATATTGGG CAACAATGCC GGTCTCGGCA

1351 CCACCAGACA TACTGTTGAA AACACCGTAT GGGGACAATC TCAAACCAAC

1401 TTTTTCATGA CCAATGGCTT CGACAAGAGC ATCAACAACT TCCAAGGTGA

1451 AACGAGCTCT GTTTTCAATA GATCCACCAT ATTCATCGGT TCTAGTATTG

1501 GAATGAGGGT CCAAGAACTG GTTTAACAAG TAACCGTTAG CACTGTGAAT

1551 TTCAACACCA TCGGCACCAG CAGCAATAGA GTTCTTGGCA GCCTGGACGT

1601 ATTCCTTAAT GTATTGCTTG ATTTCGTCCT TGGTTAGGCT GTGTTGTGGG

1651 TTGTTGGCCT TCTTGGCCTT AGCTTCTTGC TCGGCATCCA TGAAAACGTT

1701 GTCAGAAGCT GAATCGTAAC GCAAACCATC TCTGGCAAGA TTGTCTGGGA

1751 AAGCAGCCCA ACCCAAAACC CATAACTGAA CCCAAACGAA CGATTTCTTT

1801 TCATGAATAG CGTTGAAGAT TTTGGTCCAT TCCACCATTT GTTCTTCCGA

1851 CCAAACACCT GGAGCGTTAT CGTAACCGCC GGCTTGTGGG GATATGAAGG

1901 CACCTTCAGT GATAATCATG GTACCAGGTC TTTGAGCACG TTGGGTGTAG

1951 TATTCGACTG CCCAGTCCCT GTTTGGGATA TTACCAGGGT GAAGAGCTCT

2001 CATTCTGGTC AATGGAGGAA TGACAGCACG GTGCAAAAGT TCATTGTTCC

2051 CGATCTTGAT TGGTTTGAAT AGGTTGGTGT CACCTAAAGC TTGTGGCTTA

2101 AAATCTTTTA CAAATGACAT ATGTATATCT CCTTCTTAAA GTTAAACAAA

2151 ATTATTTCTA GAGGGAAACC GTTGTGGTCT CCCTATAGTG AGTCGTATTA

2201 ATTTCGCGGG ATCGAGATCT CGATCCTCTA CGCCGGACGC ATCGTGGCCG

2251 GCATCACCGG CGCCACAGGT GCGGTTGCTG GCGCCTATAT CGCCGACATC

234

2301 ACCGATGGGG AAGATCGGGC TCGCCACTTC GGGCTCATGA GCGCTTGTTT

2351 CGGCGTGGGT ATGGTGGCAG GCCCCGTGGC CGGGGGACTG TTGGGCGCCA

2401 TCTCCTTGCA TGCACCATTC CTTGCGGCGG CGGTGCTCAA CGGCCTCAAC

2451 CTACTACTGG GCTGCTTCCT AATGCAGGAG TCGCATAAGG GAGAGCGTCG

2501 ACCGATGCCC TTGAGAGCCT TCAACCCAGT CAGCTCCTTC CGGTGGGCGC

2551 GGGGCATGAC TATCGTCGCC GCACTTATGA CTGTCTTCTT TATCATGCAA

2601 CTCGTAGGAC AGGTGCCGGC AGCGCTCTGG GTCATTTTCG GCGAGGACCG

2651 CTTTCGCTGG AGCGCGACGA TGATCGGCCT GTCGCTTGCG GTATTCGGAA

2701 TCTTGCACGC CCTCGCTCAA GCCTTCGTCA CTGGTCCCGC CACCAAACGT

2751 TTCGGCGAGA AGCAGGCCAT TATCGCCGGC ATGGCGGCCG ACGCGCTGGG

2801 CTACGTCTTG CTGGCGTTCG CGACGCGAGG CTGGATGGCC TTCCCCATTA

2851 TGATTCTTCT CGCTTCCGGC GGCATCGGGA TGCCCGCGTT GCAGGCCATG

2901 CTGTCCAGGC AGGTAGATGA CGACCATCAG GGACAGCTTC AAGGATCGCT

2951 CGCGGCTCTT ACCAGCCTAA CTTCGATCAC TGGACCGCTG ATCGTCACGG

3001 CGATTTATGC CGCCTCGGCG AGCACATGGA ACGGGTTGGC ATGGATTGTA

3051 GGCGCCGCCC TATACCTTGT CTGCCTCCCC GCGTTGCGTC GCGGTGCATG

3101 GAGCCGGGCC ACCTCGACCT GAATGGAAGC CGGCGGCACC TCGCTAACGG

3151 ATTCACCACT CCAAGAATTG GAGCCAATCA ATTCTTGCGG AGAACTGTGA

3201 ATGCGCAAAC CAACCCTTGG CAGAACATAT CCATCGCGTC CGCCATCTCC

3251 AGCAGCCGCA CGCGGCGCAT CTCGGGCAGC GTTGGGTCCT GGCCACGGGT

3301 GCGCATGATC GTGCTCCTGT CGTTGAGGAC CCGGCTAGGC TGGCGGGGTT

3351 GCCTTACTGG TTAGCAGAAT GAATCACCGA TACGCGAGCG AACGTGAAGC

3401 GACTGCTGCT GCAAAACGTC TGCGACCTGA GCAACAACAT GAATGGTCTT

3451 CGGTTTCCGT GTTTCGTAAA GTCTGGAAAC GCGGAAGTCA GCGCCCTGCA

3501 CCATTATGTT CCGGATCTGC ATCGCAGGAT GCTGCTGGCT ACCCTGTGGA

3551 ACACCTACAT CTGTATTAAC GAAGCGCTGG CATTGACCCT GAGTGATTTT

3601 TCTCTGGTCC CGCCGCATCC ATACCGCCAG TTGTTTACCC TCACAACGTT

3651 CCAGTAACCG GGCATGTTCA TCATCAGTAA CCCGTATCGT GAGCATCCTC

3701 TCTCGTTTCA TCGGTATCAT TACCCCCATG AACAGAAATC CCCCTTACAC

3751 GGAGGCATCA GTGACCAAAC AGGAAAAAAC CGCCCTTAAC ATGGCCCGCT

3801 TTATCAGAAG CCAGACATTA ACGCTTCTGG AGAAACTCAA CGAGCTGGAC

3851 GCGGATGAAC AGGCAGACAT CTGTGAATCG CTTCACGACC ACGCTGATGA

3901 GCTTTACCGC AGCTGCCTCG CGCGTTTCGG TGATGACGGT GAAAACCTCT

3951 GACACATGCA GCTCCCGGAG ACGGTCACAG CTTGTCTGTA AGCGGATGCC

4001 GGGAGCAGAC AAGCCCGTCA GGGCGCGTCA GCGGGTGTTG GCGGGTGTCG

4051 GGGCGCAGCC ATGACCCAGT CACGTAGCGA TAGCGGAGTG TATACTGGCT

4101 TAACTATGCG GCATCAGAGC AGATTGTACT GAGAGTGCAC CATATATGCG

4151 GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCT

4201 CTTCCGCTTC CTCGCTCACT GACTCGCTGC GCTCGGTCGT TCGGCTGCGG

4251 CGAGCGGTAT CAGCTCACTC AAAGGCGGTA ATACGGTTAT CCACAGAATC

4301 AGGGGATAAC GCAGGAAAGA ACATGTGAGC AAAAGGCCAG CAAAAGGCCA

4351 GGAACCGTAA AAAGGCCGCG TTGCTGGCGT TTTTCCATAG GCTCCGCCCC

4401 CCTGACGAGC ATCACAAAAA TCGACGCTCA AGTCAGAGGT GGCGAAACCC

4451 GACAGGACTA TAAAGATACC AGGCGTTTCC CCCTGGAAGC TCCCTCGTGC

4501 GCTCTCCTGT TCCGACCCTG CCGCTTACCG GATACCTGTC CGCCTTTCTC

4551 CCTTCGGGAA GCGTGGCGCT TTCTCATAGC TCACGCTGTA GGTATCTCAG

4601 TTCGGTGTAG GTCGTTCGCT CCAAGCTGGG CTGTGTGCAC GAACCCCCCG

4651 TTCAGCCCGA CCGCTGCGCC TTATCCGGTA ACTATCGTCT TGAGTCCAAC

4701 CCGGTAAGAC ACGACTTATC GCCACTGGCA GCAGCCACTG GTAACAGGAT

4751 TAGCAGAGCG AGGTATGTAG GCGGTGCTAC AGAGTTCTTG AAGTGGTGGC

4801 CTAACTACGG CTACACTAGA AGGACAGTAT TTGGTATCTG CGCTCTGCTG

4851 AAGCCAGTTA CCTTCGGAAA AAGAGTTGGT AGCTCTTGAT CCGGCAAACA

235

4901 AACCACCGCT GGTAGCGGTG GTTTTTTTGT TTGCAAGCAG CAGATTACGC

4951 GCAGAAAAAA AGGATCTCAA GAAGATCCTT TGATCTTTTC TACGGGGTCT

5001 GACGCTCAGT GGAACGAAAA CTCACGTTAA GGGATTTTGG TCATGAGATT

5051 ATCAAAAAGG ATCTTCACCT AGATCCTTTT AAATTAAAAA TGAAGTTTTA

5101 AATCAATCTA AAGTATATAT GAGTAAACTT GGTCTGACAG TTACCAATGC

5151 TTAATCAGTG AGGCACCTAT CTCAGCGATC TGTCTATTTC GTTCATCCAT

5201 AGTTGCCTGA CTCCCCGTCG TGTAGATAAC TACGATACGG GAGGGCTTAC

5251 CATCTGGCCC CAGTGCTGCA ATGATACCGC GAGACCCACG CTCACCGGCT

5301 CCAGATTTAT CAGCAATAAA CCAGCCAGCC GGAAGGGCCG AGCGCAGAAG

5351 TGGTCCTGCA ACTTTATCCG CCTCCATCCA GTCTATTAAT TGTTGCCGGG

5401 AAGCTAGAGT AAGTAGTTCG CCAGTTAATA GTTTGCGCAA CGTTGTTGCC

5451 ATTGCTGCAG GCATCGTGGT GTCACGCTCG TCGTTTGGTA TGGCTTCATT

5501 CAGCTCCGGT TCCCAACGAT CAAGGCGAGT TACATGATCC CCCATGTTGT

5551 GCAAAAAAGC GGTTAGCTCC TTCGGTCCTC CGATCGTTGT CAGAAGTAAG

5601 TTGGCCGCAG TGTTATCACT CATGGTTATG GCAGCACTGC ATAATTCTCT

5651 TACTGTCATG CCATCCGTAA GATGCTTTTC TGTGACTGGT GAGTACTCAA

5701 CCAAGTCATT CTGAGAATAG TGTATGCGGC GACCGAGTTG CTCTTGCCCG

5751 GCGTCAACAC GGGATAATAC CGCGCCACAT AGCAGAACTT TAAAAGTGCT

5801 CATCATTGGA AAACGTTCTT CGGGGCGAAA ACTCTCAAGG ATCTTACCGC

5851 TGTTGAGATC CAGTTCGATG TAACCCACTC GTGCACCCAA CTGATCTTCA

5901 GCATCTTTTA CTTTCACCAG CGTTTCTGGG TGAGCAAAAA CAGGAAGGCA

5951 AAATGCCGCA AAAAAGGGAA TAAGGGCGAC ACGGAAATGT TGAATACTCA

6001 TACTCTTCCT TTTTCAATAT TATTGAAGCA TTTATCAGGG TTATTGTCTC

6051 ATGAGCGGAT ACATATTTGA ATGTATTTAG AAAAATAAAC AAATAGGGGT

6101 TCCGCGCACA TTTCCCCGAA AAGTGCCACC TGACGTCTAA GAAACCATTA

6151 TTATCATGAC ATTAACCTAT AAAAATAGGC GTATCACGAG GCCCTTTCGT

6201 CTTCAAGAA

Sequence of pBS2

1 TGGCGAATGG GACGCGCCCT GTAGCGGCGC ATTAAGCGCG GCGGGTGTGG

51 TGGTTACGCG CAGCGTGACC GCTACACTTG CCAGCGCCCT AGCGCCCGCT

101 CCTTTCGCTT TCTTCCCTTC CTTTCTCGCC ACGTTCGCCG GCTTTCCCCG

151 TCAAGCTCTA AATCGGGGGC TCCCTTTAGG GTTCCGATTT AGTGCTTTAC

201 GGCACCTCGA CCCCAAAAAA CTTGATTAGG GTGATGGTTC ACGTAGTGGG

251 CCATCGCCCT GATAGACGGT TTTTCGCCCT TTGACGTTGG AGTCCACGTT

301 CTTTAATAGT GGACTCTTGT TCCAAACTGG AACAACACTC AACCCTATCT

351 CGGTCTATTC TTTTGATTTA TAAGGGATTT TGCCGATTTC GGCCTATTGG

401 TTAAAAAATG AGCTGATTTA ACAAAAATTT AACGCGAATT TTAACAAAAT

451 ATTAACGTTT ACAATTTCAG GTGGCACTTT TCGGGGAAAT GTGCGCGGAA

501 CCCCTATTTG TTTATTTTTC TAAATACATT CAAATATGTA TCCGCTCATG

551 AGACAATAAC CCTGATAAAT GCTTCAATAA TATTGAAAAA GGAAGAGTAT

601 GAGTATTCAA CATTTCCGTG TCGCCCTTAT TCCCTTTTTT GCGGCATTTT

651 GCCTTCCTGT TTTTGCTCAC CCAGAAACGC TGGTGAAAGT AAAAGATGCT

701 GAAGATCAGT TGGGTGCACG AGTGGGTTAC ATCGAACTGG ATCTCAACAG

751 CGGTAAGATC CTTGAGAGTT TTCGCCCCGA AGAACGTTTT CCAATGATGA

801 GCACTTTTAA AGTTCTGCTA TGTGGCGCGG TATTATCCCG TATTGACGCC

851 GGGCAAGAGC AACTCGGTCG CCGCATACAC TATTCTCAGA ATGACTTGGT

901 TGAGTACTCA CCAGTCACAG AAAAGCATCT TACGGATGGC ATGACAGTAA

951 GAGAATTATG CAGTGCTGCC ATAACCATGA GTGATAACAC TGCGGCCAAC

1001 TTACTTCTGA CAACGATCGG AGGACCGAAG GAGCTAACCG CTTTTTTGCA

236

1051 CAACATGGGG GATCATGTAA CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA

1101 ATGAAGCCAT ACCAAACGAC GAGCGTGACA CCACGATGCC TGCAGCAATG

1151 GCAACAACGT TGCGCAAACT ATTAACTGGC GAACTACTTA CTCTAGCTTC

1201 CCGGCAACAA TTAATAGACT GGATGGAGGC GGATAAAGTT GCAGGACCAC

1251 TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT TTATTGCTGA TAAATCTGGA

1301 GCCGGTGAGC GTGGGTCTCG CGGTATCATT GCAGCACTGG GGCCAGATGG

1351 TAAGCCCTCC CGTATCGTAG TTATCTACAC GACGGGGAGT CAGGCAACTA

1401 TGGATGAACG AAATAGACAG ATCGCTGAGA TAGGTGCCTC ACTGATTAAG

1451 CATTGGTAAC TGTCAGACCA AGTTTACTCA TATATACTTT AGATTGATTT

1501 AAAACTTCAT TTTTAATTTA AAAGGATCTA GGTGAAGATC CTTTTTGATA

1551 ATCTCATGAC CAAAATCCCT TAACGTGAGT TTTCGTTCCA CTGAGCGTCA

1601 GACCCCGTAG AAAAGATCAA AGGATCTTCT TGAGATCCTT TTTTTCTGCG

1651 CGTAATCTGC TGCTTGCAAA CAAAAAAACC ACCGCTACCA GCGGTGGTTT

1701 GTTTGCCGGA TCAAGAGCTA CCAACTCTTT TTCCGAAGGT AACTGGCTTC

1751 AGCAGAGCGC AGATACCAAA TACTGTCCTT CTAGTGTAGC CGTAGTTAGG

1801 CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC GCTCTGCTAA

1851 TCCTGTTACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG

1901 TTGGACTCAA GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC

1951 GGGGGGTTCG TGCACACAGC CCAGCTTGGA GCGAACGACC TACACCGAAC

2001 TGAGATACCT ACAGCGTGAG CTATGAGAAA GCGCCACGCT TCCCGAAGGG

2051 AGAAAGGCGG ACAGGTATCC GGTAAGCGGC AGGGTCGGAA CAGGAGAGCG

2101 CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTTTAT AGTCCTGTCG

2151 GGTTTCGCCA CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG

2201 GGGCGGAGCC TATGGAAAAA CGCCAGCAAC GCGGCCTTTT TACGGTTCCT

2251 GGCCTTTTGC TGGCCTTTTG CTCACATGTT CTTTCCTGCG TTATCCCCTG

2301 ATTCTGTGGA TAACCGTATT ACCGCCTTTG AGTGAGCTGA TACCGCTCGC

2351 CGCAGCCGAA CGACCGAGCG CAGCGAGTCA GTGAGCGAGG AAGCGGAAGA

2401 GCGCCTGATG CGGTATTTTC TCCTTACGCA TCTGTGCGGT ATTTCACACC

2451 GCATATATGG TGCACTCTCA GTACAATCTG CTCTGATGCC GCATAGTTAA

2501 GCCAGTATAC ACTCCGCTAT CGCTACGTGA CTGGGTCATG GCTGCGCCCC

2551 GACACCCGCC AACACCCGCT GACGCGCCCT GACGGGCTTG TCTGCTCCCG

2601 GCATCCGCTT ACAGACAAGC TGTGACCGTC TCCGGGAGCT GCATGTGTCA

2651 GAGGTTTTCA CCGTCATCAC CGAAACGCGC GAGGCAGCTG CGGTAAAGCT

2701 CATCAGCGTG GTCGTGAAGC GATTCACAGA TGTCTGCCTG TTCATCCGCG

2751 TCCAGCTCGT TGAGTTTCTC CAGAAGCGTT AATGTCTGGC TTCTGATAAA

2801 GCGGGCCATG TTAAGGGCGG TTTTTTCCTG TTTGGTCACT GATGCCTCCG

2851 TGTAAGGGGG ATTTCTGTTC ATGGGGGTAA TGATACCGAT GAAACGAGAG

2901 AGGATGCTCA CGATACGGGT TACTGATGAT GAACATGCCC GGTTACTGGA

2951 ACGTTGTGAG GGTAAACAAC TGGCGGTATG GATGCGGCGG GACCAGAGAA

3001 AAATCACTCA GGGTCAATGC CAGCGCTTCG TTAATACAGA TGTAGGTGTT

3051 CCACAGGGTA GCCAGCAGCA TCCTGCGATG CAGATCCGGA ACATAATGGT

3101 GCAGGGCGCT GACTTCCGCG TTTCCAGACT TTACGAAACA CGGAAACCGA

3151 AGACCATTCA TGTTGTTGCT CAGGTCGCAG ACGTTTTGCA GCAGCAGTCG

3201 CTTCACGTTC GCTCGCGTAT CGGTGATTCA TTCTGCTAAC CAGTAAGGCA

3251 ACCCCGCCAG CCTAGCCGGG TCCTCAACGA CAGGAGCACG ATCATGCGCA

3301 CCCGTGGGGC CGCCATGCCG GCGATAATGG CCTGCTTCTC GCCGAAACGT

3351 TTGGTGGCGG GACCAGTGAC GAAGGCTTGA GCGAGGGCGT GCAAGATTCC

3401 GAATACCGCA AGCGACAGGC CGATCATCGT CGCGCTCCAG CGAAAGCGGT

3451 CCTCGCCGAA AATGACCCAG AGCGCTGCCG GCACCTGTCC TACGAGTTGC

3501 ATGATAAAGA AGACAGTCAT AAGTGCGGCG ACGATAGTCA TGCCCCGCGC

3551 CCACCGGAAG GAGCTGACTG GGTTGAAGGC TCTCAAGGGC ATCGGTCGAG

3601 ATCCCGGTGC CTAATGAGTG AGCTAACTTA CATTAATTGC GTTGCGCTCA

237

3651 CTGCCCGCTT TCCAGTCGGG AAACCTGTCG TGCCAGCTGC ATTAATGAAT

3701 CGGCCAACGC GCGGGGAGAG GCGGTTTGCG TATTGGGCGC CAGGGTGGTT

3751 TTTCTTTTCA CCAGTGAGAC GGGCAACAGC TGATTGCCCT TCACCGCCTG

3801 GCCCTGAGAG AGTTGCAGCA AGCGGTCCAC GCTGGTTTGC CCCAGCAGGC

3851 GAAAATCCTG TTTGATGGTG GTTAACGGCG GGATATAACA TGAGCTGTCT

3901 TCGGTATCGT CGTATCCCAC TACCGAGATA TCCGCACCAA CGCGCAGCCC

3951 GGACTCGGTA ATGGCGCGCA TTGCGCCCAG CGCCATCTGA TCGTTGGCAA

4001 CCAGCATCGC AGTGGGAACG ATGCCCTCAT TCAGCATTTG CATGGTTTGT

4051 TGAAAACCGG ACATGGCACT CCAGTCGCCT TCCCGTTCCG CTATCGGCTG

4101 AATTTGATTG CGAGTGAGAT ATTTATGCCA GCCAGCCAGA CGCAGACGCG

4151 CCGAGACAGA ACTTAATGGG CCCGCTAACA GCGCGATTTG CTGGTGACCC

4201 AATGCGACCA GATGCTCCAC GCCCAGTCGC GTACCGTCTT CATGGGAGAA

4251 AATAATACTG TTGATGGGTG TCTGGTCAGA GACATCAAGA AATAACGCCG

4301 GAACATTAGT GCAGGCAGCT TCCACAGCAA TGGCATCCTG GTCATCCAGC

4351 GGATAGTTAA TGATCAGCCC ACTGACGCGT TGCGCGAGAA GATTGTGCAC

4401 CGCCGCTTTA CAGGCTTCGA CGCCGCTTCG TTCTACCATC GACACCACCA

4451 CGCTGGCACC CAGTTGATCG GCGCGAGATT TAATCGCCGC GACAATTTGC

4501 GACGGCGCGT GCAGGGCCAG ACTGGAGGTG GCAACGCCAA TCAGCAACGA

4551 CTGTTTGCCC GCCAGTTGTT GTGCCACGCG GTTGGGAATG TAATTCAGCT

4601 CCGCCATCGC CGCTTCCACT TTTTCCCGCG TTTTCGCAGA AACGTGGCTG

4651 GCCTGGTTCA CCACGCGGGA AACGGTCTGA TAAGAGACAC CGGCATACTC

4701 TGCGACATCG TATAACGTTA CTGGTTTCAC ATTCACCACC CTGAATTGAC

4751 TCTCTTCCGG GCGCTATCAT GCCATACCGC GAAAGGTTTT GCGCCATTCG

4801 ATGGTGTCCG GGATCTCGAC GCTCTCCCTT ATGCGACTCC TGCATTAGGA

4851 AGCAGCCCAG TAGTAGGTTG AGGCCGTTGA GCACCGCCGC CGCAAGGAAT

4901 GGTGCATGCA AGGAGATGGC GCCCAACAGT CCCCCGGCCA CGGGGCCTGC

4951 CACCATACCC ACGCCGAAAC AAGCGCTCAT GAGCCCGAAG TGGCGAGCCC

5001 GATCTTCCCC ATCGGTGATG TCGGCGATAT AGGCGCCAGC AACCGCACCT

5051 GTGGCGCCGG TGATGCCGGC CACGATGCGT CCGGCGTAGA GGATCGAGAT

5101 CTCGATCCCG CGAAATTAAT ACGACTCACT ATAGGGGAAT TGTGAGCGGA

5151 TAACAATTCC CCTCTAGAAA TAATTTTGTT TAACTTTAAG AAGGAGATAT

5201 ACATAATGAC CAAGTTACCT ATACTAGGTT ATTGGAAAAT TAAGGGCCTT

5251 GTGCAACCCA CTCGACTTCT TTTGGAATAT CTTGAAGAAA AATATGAAGA

5301 GCATTTGTAT GAGCGCGATG AAGGTGATAA ATGGCGAAAC AAAAAGTTTG

5351 AATTGGGTTT GGAGTTTCCC AATCTTCCTT ATTATATTGA TGGTGATGTT

5401 AAATTAACAC AGTCTATGGC CATCATACGT TATATAGCTG ACAAGCACAA

5451 CATGTTGGGT GGTTGTCCAA AAGAGCGTGC AGAGATTTCA ATGCTTGAAG

5501 GAGCGGTTTT GGATATTAGA TACGGTGTTT CGAGAATTGC ATATAGTAAA

5551 GACTTTGAAA CTCTCAAAGT TGATTTTCTT AGCAAGCTAC CTGAAATGCT

5601 GAAAATGTTC GAAGATCGTT TATGTCATAA AACATATTTA AATGGTGATC

5651 ATGTAACCCA TCCTGACTTC ATGTTGTATG ACGCTCTTGA TGTTGTTTTA

5701 TACATGGACC CAATGTGCCT GGATGCGTTC CCAAAATTAG TTTGTTTTAA

5751 AAAACGTATT GAAGCTATCC CACAAATTGA TAAGTACTTG AAATCCAGCA

5801 AGTATATAGC ATGGCCTTTG CAGGGCTGGC AAGCCACGTT TGGTGGTGGC

5851 GACCATCCTC CAAAATCGGA TCATCTGGTT CCGCGTCATA TGCCCATGTC

5901 TTCAGTCAAA ATTTCTCCAT TGAAGGATTC TGAAGCATTC CAGTCTATCA

5951 AAGTTGGTAA CAACACTCTT CAAACCAAGA TTGTCTATCC ACCAACTACT

6001 AGATTTAGAG CTTTAGAAGA CCACACTCCT TCTGATTTGC AATTGCAGTA

6051 CTATGGCGAC AGATCCACTT TCCCAGGTAC TTTGCTTATC ACTGAAGCTA

6101 CTTTTGTCTC TCCTCAAGCC TCTGGTTATG AAGGTGCTGC TCCAGGTATT

6151 TGGACTGACA AGCACGCTAA AGCATGGAAG GTTATTACTG ATAAAGTTCA

6201 TGCCAACGGT TCTTTCGTTT CAACCCAGTT GATTTTTTTG GGAAGGGTTG

238

6251 CAGATCCAGC TGTTATGAAG ACCCGTGGGT TGAATCCAGT TTCTGCCTCT

6301 GCTACTTATG AAAGTGATGC CGCTAAAGAA GCTGCCGAAG CAGTTGGTAA

6351 CCCTGTTAGA GCTTTGACTA CCCAAGAAGT CAAGGATCTT GTTTACGAGG

6401 CTTACACCAA CGCTGCTCAG AAGGCCATGG ATGCTGGTTT CGACTATATT

6451 GAACTCCATG CTGCTCATGG CTACCTTTTA GATCAATTTT TGCAACCATG

6501 CACCAATCAA AGAACTGATG AATACGGTGG ATCCATTGAG AACAGAGCCA

6551 GGTTAATTCT TGAGTTGATT GACCATTTGT CTACCATTGT CGGTGCTGAC

6601 AAGATTGGTA TCAGAATCTC TCCATGGGCT ACTTTCCAAA ACATGAAGGC

6651 TCACAAGGAC ACTGTTCACC CATTGACTAC TTTCTCTTAC TTGGTCCACG

6701 AATTGCAACA GAGAGCTGAC AAGGGTCAAG GTATTGCCTA CATTTCTGTC

6751 GTTGAGCCTC GTGTAAGTGG TAACGTCGAC GTCTCTGAAG AAGACCAAGC

6801 TGGTGACAAC GAATTTGTCT CCAAGATCTG GAAGGGTGTT ATCTTGAAGG

6851 CAGGTAACTA CTCCTACGAT GCTCCAGAGT TCAAGACATT GAAGGAAGAT

6901 ATCGCTGACA AGCGTACATT AGTTGGCTTC TCCAGATACT TCACCTCGAA

6951 TCCTAACTTG GTTTGGAAAT TGCGTGATGG AATTGACTTG GTGCCATACG

7001 ACAGAAACAC GTTCTACAGT GACAATAACT ATGGTTACAA TACCTTTTCT

7051 ATGGATTCCG AAGAGGTTGA TAAAGAATTA GAAATCAAGA GAGTTCCTTC

7101 GGCCATTGAA GCTTTGTGAT GCGGCCGCAC TCGAGCACCA CCACCACCAC

7151 CACTGAGATC CGGCTGCTAA CAAAGCCCGA AAGGAAGCTG AGTTGGCTGC

7201 TGCCACCGCT GAGCAATAAC TAGCATAACC CCTTGGGGCC TCTAAACGGG

7251 TCTTGAGGGG TTTTTTGCTG AAAGGAGGAA CTATATCCGG AT

Sequence of pFB1




























239





















































240


























5201 ACATATGCCC ATGTCTTCAG TCAAAATTTC TCCATTGAAG GATTCTGAAG

5251 CATTCCAGTC TATCAAAGTT GGTAACAACA CTCTTCAAAC CAAGATTGTC

5301 TATCCACCAA CTACTAGATT TAGAGCTTTA GAAGACCACA CTCCTTCTGA

5351 TTTGCAATTG CAGTACTATG GCGACAGATC CACTTTCCCA GGTACTTTGC

5401 TTATCACTGA AGCTACTTTT GTCTCTCCTC AAGCCTCTGG TTATGAAGGT

5451 GCTGCTCCAG GTATTTGGAC TGACAAGCAC GCTAAAGCAT GGAAGGTTAT

5501 TACTGATAAA GTTCATGCCA ACGGTTCTTT CGTTTCAACC CAGTTGATTT

5551 TTTTGGGAAG GGTTGCAGAT CCAGCTGTTA TGAAGACCCG TGGGTTGAAT

5601 CCAGTTTCTG CCTCTGCTAC TTATGAAAGT GATGCCGCTA AAGAAGCTGC

5651 CGAAGCAGTT GGTAACCCTG TTAGAGCTTT GACTACCCAA GAAGTCAAGG

5701 ATCTTGTTTA CGAGGCTTAC ACCAACGCTG CTCAGAAGGC CATGGATGCT

5751 GGTTTCGACT ATATTGAACT CCATGCTGCT CATGGCTACC TTTTAGATCA

5801 ATTTTTGCAA CCATGCACCA ATCAAAGAAC TGATGAATAC GGTGGATCCA

5851 TTGAGAACAG AGCCAGGTTA ATTCTTGAGT TGATTGACCA TTTGTCTACC

5901 ATTGTCGGTG CTGACAAGAT TGGTATCAGA ATCTCTCCAT GGGCTACTTT

5951 CCAAAACATG AAGGCTCACA AGGACACTGT TCACCCATTG ACTACTTTCT

6001 CTTACTTGGT CCACGAATTG CAACAGAGAG CTGACAAGGG TCAAGGTATT

6051 GCCTACATTT CTGTCGTTGA GCCTCGTGTA AGTGGTAACG TCGACGTCTC

6101 TGAAGAAGAC CAAGCTGGTG ACAACGAATT TGTCTCCAAG ATCTGGAAGG

6151 GTGTTATCTT GAAGGCAGGT AACTACTCCT ACGATGCTCC AGAGTTCAAG

6201 ACATTGAAGG AAGATATCGC TGACAAGCGT ACATTAGTTG GCTTCTCCAG

6251 ATACTTCACC TCGAATCCTA ACTTGGTTTG GAAATTGCGT GATGGAATTG

6301 ACTTGGTGCC ATACGACAGA AACACGTTCT ACAGTGACAA TAACTATGGT

6351 TACAATACCT TTTCTATGGA TTCCGAAGAG GTTGATAAAG AATTAGAAAT

6401 CAAGAGAGTT CCTTCGGCCA TTGAAGCTTT GTGATGCGGC CGCACTCGAG

6451 CACCACCACC ACCACCACTG AGATCCGGCT GCTAACAAAG CCCGAAAGGA

6501 AGCTGAGTTG GCTGCTGCCA CCGCTGAGCA ATAACTAGCA TAACCCCTTG

241

6551 GGGCCTCTAA ACGGGTCTTG AGGGGTTTTT TGCTGAAAGG AGGAACTATA

6601 TCCGGAT

Sequence of pRP4















































242





















































243







5201 ACATATGCCA TTTGTAAAAG GTTTTGAGCC GATCTCCCTA AGAGACACAA

5251 ACCTTTTTGA ACCAATTAAG ATTGGTAACA CTCAGCTTGC ACATCGTGCG

5301 GTTATGCCCC CATTGACCAG AATGAGGGCC ACTCACCCCG GAAATATTCC

5351 AAATAAGGAG TGGGCTGCTG TGTATTATGG TCAGCGTGCT CAAAGACCTG

5401 GTACCATGAT CATCACGGAA GGTACGTTTA TTTCCCCTCA AGCCGGCGGC

5451 TATGACAACG CCCCTGGGAT TTGGTCTGAT GAGCAGGTCG CTGAGTGGAA

5501 GAATATCTTT TTAGCCATCC ATGATTGTCA GTCGTTCGCG TGGGTACAAC

5551 TTTGGTCTTT AGGCTGGGCA TCCTTCCCAG ACGTATTGGC AAGAGACGGG

5601 TTACGCTATG ACTGTGCATC TGACAGAGTG TATATGAATG CTACGTTACA

5651 AGAAAAGGCC AAAGATGCGA ATAATCTCGA ACATAGTTTG ACTAAAGACG

5701 ACATTAAACA GTATATCAAG GATTACATCC ATGCGGCTAA GAATTCTATC

5751 GCGGCTGGCG CCGATGGTGT AGAAATTCAT AGCGCCAATG GGTACTTGTT

5801 GAATCAGTTC TTGGATCCAC ATTCTAATAA GAGGACCGAC GAATACGGCG

5851 GAACGATCGA AAACAGGGCC CGCTTTACAC TGGAGGTTGT CGATGCTCTT

5901 ATCGAAACTA TCGGTCCTGA ACGGGTGGGT TTGAGGTTGT CGCCGTACGG

5951 CACTTTTAAC AGTATGTCTG GGGGTGCTGA ACCAGGTATT ATCGCTCAAT

6001 ATTCGTATGT TTTGGGTGAA TTAGAGAAGA GGGCAAAGGC TGGTAAGCGT

6051 TTGGCCTTTG TGCACCTCGT TGAACCACGT GTCACGGACC CATCGTTGGT

6101 GGAGGGCGAA GGAGAATATT CCGAGGGTAC TAACGATTTT GCCTACTCTA

6151 TATGGAAGGG TCCAATCATC AGAGCTGGTA ATTACGCTCT TCATCCAGAA

6201 GTGGTTAGAG AACAAGTAAA GGATCCCAGA ACCTTGATAG GCTATGGTAG

6251 ATTCTTCATC TCTAACCCAG ATTTAGTCTA CCGTTTAGAA GAGGGCCTGC

6301 CATTGAACAA GTATGACAGA AGTACCTTCT ACACCATGTC CGCGGAAGGT

6351 TATACCGACT ACCCAACATA CGAAGAGGCA GTAGATTTAG GTTGGAACAA

6401 GAACTGATGC GGCCGCACTC GAGCACCACC ACCACCACCA CTGAGATCCG

6451 GCTGCTAACA AAGCCCGAAA GGAAGCTGAG TTGGCTGCTG CCACCGCTGA

6501 GCAATAACTA GCATAACCCC TTGGGGCCTC TAAACGGGTC TTGAGGGGTT

6551 TTTTGCTGAA AGGAGGAACT ATATCCGGAT

Sequence of pGF23

1 TTCTCATGTT TGACAGCTTA TCATCGATAA GCTTTGGAGA TTATCGTCAC

51 TGCAATGCTT CGCAATATGG CGCAAAATGA CCAACAGCGG TTGATTGATC

101 AGGTAGAGGG GGCGCTGTAC GAGGTAAAGC CCGATGCCAG CATTCCTGAC

151 GACGATACGG AGCTGCTGCG CGATTACGTA AAGAAGTTAT TGAAGCATCC

201 TCGTCAGTAA AAAGTTAATC TTTTCAACAG CTGTCATAAA GTTGTCACGG

251 CCGAGACTTA TAGTCGCTTT GTTTTTATTT TTTAATGTAT TTGTACATGG

301 AGAAAATAAA GTGAAACAGT CGACTGACAG GAAGAGCAAG ATTGTGCAGA

351 GGGCAGCTCC AGAAGTTCAA GAGGATGTCA AGACTTTCAA GACAGACCTG

401 CTGAGCACCA TGGATTCAAC CACCCGAAGC CATTCATTTC CTAGTTTCCA

451 GGAGCCAGAG CAGACTGAAG GGGCAGTTCC CCACCTGATT CAGAACAATA

501 TGACTGGAAG CCAGGCTTTC GGTTATGACC AATTTTTCAG AGACAAGATC

551 ATGGAGAAGA AACAGGACCA CACCTACCGT GTGTTCAAGA CTGTGAATCG

601 TTGGGCTAAT GCCTACCCCT TTGCCCAACA CTTCTCCGAG GCATCTATGG

651 CATCAAAGGA TGTTTCTGTT TGGTGTAGTA ATGACTATTT GGGCATAAGC

244

701 AGACACCCTC GTGTCTTGCA GGCCATAGAG GAGACCCTGA AGAATCATGG

751 AGCTGGAGCT GGGGGCACTC GCAATATCTC AGGTACCAGC AAGTTTCATG

801 TGGAGCTTGA ACAGGAGCTG GCTGAACTAC ACCAGAAAGA CTCAGCTCTG

851 CTCTTCTCCT CCTGTTTTGT GGCCAATGAT TCTACTCTCT TTACACTGGC

901 CAAGCTTCTG CCAGGGTGTG AGATCTACTC AGATGCAGGC AATCATGCCT

951 CCATGATCCA AGGCATTCGC AACAGTGGTG CAGCCAAGTT TGTCTTCAGA

1001 CACAATGACC CAGGCCACCT GAAGAAACTT CTCGAGAAGT CTGATCCCAA

1051 GACACCAAAA ATTGTGGCTT TTGAGACTGT TCATTCCATG GATGGTGCCA

1101 TCTGTCCTCT GGAGGAATTG TGTGATGTGG CCCACCAGTA TGGAGCCCTG

1151 ACCTTCGTAG ATGAAGTCCA TGCTGTAGGA CTGTATGGAG CCCGGGGTGC

1201 AGGTATCGGG GAGCGTGATG GAATTATGCA CAAGCTTGAC ATCATCTCTG

1251 GAACTCTTGG CAAGGCCTTT GGTTGCGTCG GTGGCTATAT AGCCAGCACT

1301 CGGGACTTGG TGGACATGGT GCGCTCCTAC GCTGCAGGCT TCATCTTTAC

1351 CACTTCACTG CCTCCCATGA TGCTCTCTGG GGCTCTAGAA TCTGTGCGCC

1401 TACTCAAGGG AGAGGAGGGT CAAGCCCTGA GGCGGGCACA CCAGCGCAAT

1451 GTCAAACACA TGCGCCAGCT GCTAATGGAC AGGGGCTTTC CTGTTATCCC

1501 CTGTCCCAGC CACATCATCC CCATCAGGGT GGGTAATGCA GCACTCAACA

1551 GCAAGATCTG TGATCTTCTG CTCTCCAAGC ACAGCATCTA TGTGCAGGCC

1601 ATCAACTACC CAACTGTGCC TCGTGGTGAG GAGCTACTGC GCTTGGCCCC

1651 CTCCCCCCAC CACAGCCCTC AGATGATGGA AAACTTTGTG GAGAAGCTGC

1701 TGCTGGCCTG GACTGAGGTG GGGCTGCCCC TCCAAGATGT GTCTGTGGCT

1751 GCATGCAACT TCTGTCATCG TCCTGTGCAC TTTGAACTTA TGAGCGAGTG

1801 GGAGCGATCC TACTTTGGGA ACATGGGACC CCAATATGTT ACCACCTATG

1851 CTTAAGGGGA TCCTCTACGC CGGACGCATC GTGGCCGGCA TCACCGGCGC

1901 CACAGGTGCG GTTGCTGGCG CCTATATCGC CGACATCACC GATGGGGAAG

1951 ATCGGGCTCG CCACTTCGGG CTCATGAGCG CTTGTTTCGG CGTGGGTATG

2001 GTGGCAGGCC CCGTGGCCGG GGGACTGTTG GGCGCCATCT CCTTGCATGC

2051 ACCATTCCTT GCGGCGGCGG TGCTCAACGG CCTCAACCTA CTACTGGGCT

2101 GCTTCCTAAT GCAGGAGTCG CATAAGGGAG AGCGTCGATC GACCGATGCC

2151 CTTGAGAGCC TTCAACCCAG TCAGCTCCTT CCGGTGGGCG CGGGGCATGA

2201 CTATCGTCGC CGCACTTATG ACTGTCTTCT TTATCATGCA ACTCGTAGGA

2251 CAGGTGCCGG CAGCGCTCTG GGTCATTTTC GGCGAGGACC GCTTTCGCTG

2301 GAGCGCGACG ATGATCGGCC TGTCGCTTGC GGTATTCGGA ATCTTGCACG

2351 CCCTCGCTCA AGCCTTCGTC ACTGGTCCCG CCACCAAACG TTTCGGCGAG

2401 AAGCAGGCCA TTATCGCCGG CATGGCGGCC GACGCGCTGG GCTACGTCTT

2451 GCTGGCGTTC GCGACGCGAG GCTGGATGGC CTTCCCCATT ATGATTCTTC

2501 TCGCTTCCGG CGGCATCGGG ATGCCCGCGT TGCAGGCCAT GCTGTCCAGG

2551 CAGGTAGATG ACGACCATCA GGGACAGCTT CAAGGATCGC TCGCGGCTCT

2601 TACCAGCCTA ACTTCGATCA TTGGACCGCT GATCGTCACG GCGATTTATG

2651 CCGCCTCGGC GAGCACATGG AACGGGTTGG CATGGATTGT AGGCGCCGCC

2701 CTATACCTTG TCTGCCTCCC CGCGTTGCGT CGCGGTGCAT GGAGCCGGGC

2751 CACCTCGACC TGAATGGAAG CCGGCGGCAC CTCGCTAACG GATTCACCAC

2801 TCCAAGAATT GGAGCCAATC AATTCTTGCG GAGAACTGTG AATGCGCAAA

2851 CCAACCCTTG GCAGAACATA TCCATCGCGT CCGCCATCTC CAGCAGCCGC

2901 ACGCGGCGCA TCTCGGGCAG CGTTGGGTCC TGGCCACGGG TGCGCATGAT

2951 CGTGCTCCTG TCGTTGAGGA CCCGGCTAGG CTGGCGGGGT TGCCTTACTG

3001 GTTAGCAGAA TGAATCACCG ATACGCGAGC GAACGTGAAG CGACTGCTGC

3051 TGCAAAACGT CTGCGACCTG AGCAACAACA TGAATGGTCT TCGGTTTCCG

3101 TGTTTCGTAA AGTCTGGAAA CGCGGAAGTC AGCGCCCTGC ACCATTATGT

3151 TCCGGATCTG CATCGCAGGA TGCTGCTGGC TACCCTGTGG AACACCTACA

3201 TCTGTATTAA CGAAGCGCTG GCATTGACCC TGAGTGATTT TTCTCTGGTC

3251 CCGCCGCATC CATACCGCCA GTTGTTTACC CTCACAACGT TCCAGTAACC

245

3301 GGGCATGTTC ATCATCAGTA ACCCGTATCG TGAGCATCCT CTCTCGTTTC

3351 ATCGGTATCA TTACCCCCAT GAACAGAAAT CCCCCTTACA CGGAGGCATC

3401 AGTGACCAAA CAGGAAAAAA CCGCCCTTAA CATGGCCCGC TTTATCAGAA

3451 GCCAGACATT AACGCTTCTG GAGAAACTCA ACGAGCTGGA CGCGGATGAA

3501 CAGGCAGACA TCTGTGAATC GCTTCACGAC CACGCTGATG AGCTTTACCG

3551 CAGCTGCCTC GCGCGTTTCG GTGATGACGG TGAAAACCTC TGACACATGC

3601 AGCTCCCGGA GACGGTCACA GCTTGTCTGT AAGCGGATGC CGGGAGCAGA

3651 CAAGCCCGTC AGGGCGCGTC AGCGGGTGTT GGCGGGTGTC GGGGCGCAGC

3701 CATGACCCAG TCACGTAGCG ATAGCGGAGT GTATACTGGC TTAACTATGC

3751 GGCATCAGAG CAGATTGTAC TGAGAGTGCA CCATATGCGG TGTGAAATAC

3801 CGCACAGATG CGTAAGGAGA AAATACCGCA TCAGGCGCTC TTCCGCTTCC

3851 TCGCTCACTG ACTCGCTGCG CTCGGTCGTT CGGCTGCGGC GAGCGGTATC

3901 AGCTCACTCA AAGGCGGTAA TACGGTTATC CACAGAATCA GGGGATAACG

3951 CAGGAAAGAA CATGTGAGCA AAAGGCCAGC AAAAGGCCAG GAACCGTAAA

4001 AAGGCCGCGT TGCTGGCGTT TTTCCATAGG CTCCGCCCCC CTGACGAGCA

4051 TCACAAAAAT CGACGCTCAA GTCAGAGGTG GCGAAACCCG ACAGGACTAT

4101 AAAGATACCA GGCGTTTCCC CCTGGAAGCT CCCTCGTGCG CTCTCCTGTT

4151 CCGACCCTGC CGCTTACCGG ATACCTGTCC GCCTTTCTCC CTTCGGGAAG

4201 CGTGGCGCTT TCTCATAGCT CACGCTGTAG GTATCTCAGT TCGGTGTAGG

4251 TCGTTCGCTC CAAGCTGGGC TGTGTGCACG AACCCCCCGT TCAGCCCGAC

4301 CGCTGCGCCT TATCCGGTAA CTATCGTCTT GAGTCCAACC CGGTAAGACA

4351 CGACTTATCG CCACTGGCAG CAGCCACTGG TAACAGGATT AGCAGAGCGA

4401 GGTATGTAGG CGGTGCTACA GAGTTCTTGA AGTGGTGGCC TAACTACGGC

4451 TACACTAGAA GGACAGTATT TGGTATCTGC GCTCTGCTGA AGCCAGTTAC

4501 CTTCGGAAAA AGAGTTGGTA GCTCTTGATC CGGCAAACAA ACCACCGCTG

4551 GTAGCGGTGG TTTTTTTGTT TGCAAGCAGC AGATTACGCG CAGAAAAAAA

4601 GGATCTCAAG AAGATCCTTT GATCTTTTCT ACGGGGTCTG ACGCTCAGTG

4651 GAACGAAAAC TCACGTTAAG GGATTTTGGT CATGAGATTA TCAAAAAGGA

4701 TCTTCACCTA GATCCTTTTA AATTAAAAAT GAAGTTTTAA ATCAATCTAA

4751 AGTATATATG AGTAAACTTG GTCTGACAGT TACCAATGCT TAATCAGTGA

4801 GGCACCTATC TCAGCGATCT GTCTATTTCG TTCATCCATA GTTGCCTGAC

4851 TCCCCGTCGT GTAGATAACT ACGATACGGG AGGGCTTACC ATCTGGCCCC

4901 AGTGCTGCAA TGATACCGCG AGACCCACGC TCACCGGCTC CAGATTTATC

4951 AGCAATAAAC CAGCCAGCCG GAAGGGCCGA GCGCAGAAGT GGTCCTGCAA

5001 CTTTATCCGC CTCCATCCAG TCTATTAATT GTTGCCGGGA AGCTAGAGTA

5051 AGTAGTTCGC CAGTTAATAG TTTGCGCAAC GTTGTTGCCA TTGCTGCAGG

5101 CATCGTGGTG TCACGCTCGT CGTTTGGTAT GGCTTCATTC AGCTCCGGTT

5151 CCCAACGATC AAGGCGAGTT ACATGATCCC CCATGTTGTG CAAAAAAGCG

5201 GTTAGCTCCT TCGGTCCTCC GATCGTTGTC AGAAGTAAGT TGGCCGCAGT

5251 GTTATCACTC ATGGTTATGG CAGCACTGCA TAATTCTCTT ACTGTCATGC

5301 CATCCGTAAG ATGCTTTTCT GTGACTGGTG AGTACTCAAC CAAGTCATTC

5351 TGAGAATAGT GTATGCGGCG ACCGAGTTGC TCTTGCCCGG CGTCAACACG

5401 GGATAATACC GCGCCACATA GCAGAACTTT AAAAGTGCTC ATCATTGGAA

5451 AACGTTCTTC GGGGCGAAAA CTCTCAAGGA TCTTACCGCT GTTGAGATCC

5501 AGTTCGATGT AACCCACTCG TGCACCCAAC TGATCTTCAG CATCTTTTAC

5551 TTTCACCAGC GTTTCTGGGT GAGCAAAAAC AGGAAGGCAA AATGCCGCAA

5601 AAAAGGGAAT AAGGGCGACA CGGAAATGTT GAATACTCAT ACTCTTCCTT

5651 TTTCAATATT ATTGAAGCAT TTATCAGGGT TATTGTCTCA TGAGCGGATA

5701 CATATTTGAA TGTATTTAGA AAAATAAACA AATAGGGGTT CCGCGCACAT

5751 TTCCCCGAAA AGTGCCACCT GACGTCTAAG AAACCATTAT TATCATGACA

5801 TTAACCTATA AAAATAGGCG TATCACGAGG CCCTTTCGTC TTCAAGAA

246

APPENDIX D GC AND HPLC METHODS

AZW2.Meth

Figure D-1. AZW2.Meth began with an initial temperature of 140°C for 10 min, followed by an increase at 20°C/min to a temperature of 180°C at which the program remained for 3 min.

AZW3.Meth

Figure D-2. AZW3.Meth began with an initial temperature of 100°C for 12 min, followed by an increase at 20°C/min to a temperature of 180°C at which the program remained for 5 min.

247

BTS2.Meth

Figure D-3. BTS2.Meth began with an initial temperature of 100°C for 10 min, followed by an increase at 20°C/min to a temperature of 180°C at which the program remained for 3 min.

BTS3.Meth


248

BTS4.Meth

Figure D-5. BTS4.Meth began with an initial temperature of 70°C for 2 min, followed by an increase at 0.3°C/min to a temperature of 90°C which then immediately increased at 20°C/min to temperature of 180°C at which the program remained for 3 min.

BTS7.Meth


249

BTS8.Meth


FB1.Meth

Figure D-8. FB1.Meth began with an initial temperature of 60°C for 5 min, followed by an increase at 20°C/min to a temperature of 200°C at which the program remained for 8 min.

250

JON.Meth

Figure D-9. JON.Meth began with an initial temperature of 60°C for 2 min, followed by an increase at 10°C/min to a temperature of 195°C at which the program remained for 10 min.

SEF.Meth

Figure D-10. SEF.Meth began with an initial temperature of 60°C for 5 min, followed by an increase at 10°C/min to a temperature of 195°C at which the program remained for 10 min.

251

YAP.Meth

Figure D-11. YAP.Meth began with an initial temperature of 90°C, followed by an increase at 10°C/min to a temperature of 130°C, followed by an increase at 2°C/min to a temperature of 150°C, followed by an increase at a rate of 20°C/min to a temperature of 250°C at which the program remained for 5 min.

LMM.Meth

Figure D-12. LMM.Meth had a flow rate of 1 mL/min and began with initial conditions of 3% B which were maintained for 2.5 min. This was followed by a linear increase to 18% B over 5 min which was then followed by a linear increase to 28% B over 3.5 min. Next followed a linear increase to 90% B over 10 min. After a 5 min hold at 90% B, a linear decrease to 3% B over 3 min was followed by a 5 min hold at the initial conditions of 3% B.

252

RWP2.Meth

Figure D-13. RWP2.Meth had a flow rate of 1 mL/min and with initial conditions of 0 % B. The method began with an immediate linear increase to 15% B over 15 min, followed by an another increase to 50% B over 15 min, followed by yet another linear increase to 100% B over 4 min at which the program stayed for 3 min. This was followed by a linear decrease to 0% B over 3 min at which the program remained for 10 min.

253

APPENDIX E PLASMID MAPS

pET3b-OYE

Figure E-1. pET3b-OYE.

pBS2

Figure E-2. pBS2.

pET3b-OYE6209 bp

T7 terminator - 404

NdeI - 549 - CA'TA_TG

Trp 116 - 1770


T7 promoter - 2185

T7 te rminator T7 tag

OY

E

T7 p

r omo t er

bla

pBS27292 bp

T7 term - 72

XhoI - 158 - C'TCGA_G


XbaI - 2125 - T'CTAG_A

T7 promoter - 2177

OYE2.6-GS

T Fusio

n P

rote

in

bla

T7 term

T 7 pro

mot

e r

254

pFB1

Figure E-3. pFB1.

pRP4

Figure E-4. pRP4.

pFBI6607 bp

T7 term - 26



T7 promoter - 1474

OYE2.6bla

T7 promo ter

T7 term

pRP46580 bp

T7 term - 26



T7 promotor - 1447

bla

OYE3

T7 term

T 7 promotor

255

pGF23

Figure E-5. pGF23.

pGF235848 bp

pho A promoter - 229

SalI - 319 - G'TCGA_C

BamHI - 1858 - G'GATC_Cm

AL

AS

bla

pho promoter

256

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BIOGRAPHICAL SKETCH

Robert Wilson Powell III was born in Pensacola Florida in November 28, 1982.

He graduated from Florida A & M University in 2010 with a B.Sc. in Chemistry. He

began studying at the University of Florida in 2011. There he joined Dr. Jon Stewart’s

group and began studying biocatalysis. After which, he spent the next five years running

the reactions that are mentioned in this document.

© 2016 Robert Wilson Powell III

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