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Discovery and engineering

of redox enzymes

Marco Fraaije

Molecular Enzymology

University of Groningen

m.w.fraaije@rug.nl

www.gecco-biotech.com

HORIZON 2020 SUMMER SCHOOL 2017

Biocatalysis as a Key Enabling Technology

CarbaZymes / ROBOX / Metafluidics

EU Horizon 2020 Innovation Action “ROBOX”

Expanding the industrial use of Robust Oxidative Biocatalysts for the conversion and production of alcohols.

“The research for this work has received funding from the European Union (EU) project ROBOX (grant agreement n°635734) under EU’s Horizon 2020 Programme Research and Innovation actions

H2020-LEIT BIO-2014-1. Any statement made herein reflects only the author’s views. The European Union is not liable for any use that may be made of the information contained herein.”

flavoenzymes: versatile biocats

see Youtube for a movie on enzyme engineering:

search for ‘enzyme’ and ‘robox’

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focus on flavin-dependent enzymes

why?

flavins are the most versatile cofactors

oxidation

reduction light sensinge- transporthydrolysis

halogenationlight production

magnetosensing

and more...

focus on flavin-dependent enzymes

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flavoenzymes: chemically versatile

.

e-

e-

2e-

H2O2

H2O

O2

P

Preductase

P

dehydrogenaseP

oxidase

luciferase

light sensing

e- transport

magnetosensing

PPmonooxygenase

Alditol oxidase

Putrescine oxidase

Chitooligosaccharide oxidase

Eugenol oxidase

Hydroxymethylfurfural oxidase

Cytokinin dehydrogenase

Galactonolactone dehydrogenase

Phenylacetone monooxygenase

Styrene monooxygenase

Trimethylamine monooxygenase

Cyclohexanone monooxygenase

Ethionamide monooxygenase

Steroid monooxygenase

4-hydroxyacetophenone monooxygenase

O2

O2

O2

O2

CytC

NADPH

O2

NADPH

O2

NADPH

O2

NADPH

O2

NADPH

O2

NADPH

O2

NADPH

O2

Q

O2

O2

enzymes discovered @ RUG

flavoproteinoxidases

flavoproteinmonooxygenases

flavoproteindehydrogenases

flavoenzymes: versatile biocats

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Alditol oxidase

Putrescine oxidase

Chitooligosaccharide oxidase

Eugenol oxidase

Hydroxymethylfurfural oxidase

Cytokinin dehydrogenase

Galactonolactone dehydrogenase

Phenylacetone monooxygenase

Styrene monooxygenase

Trimethylamine monooxygenase

Cyclohexanone monooxygenase

Ethionamide monooxygenase

Steroid monooxygenase

4-hydroxyacetophenone monooxygenase

O2

O2

O2

O2

CytC

NADPH

O2

NADPH

O2

NADPH

O2

NADPH

O2

NADPH

O2

NADPH

O2

NADPH

O2

Q

O2

O2

oxidasesenzymes discovered @ RUG

Tapping the power of oxidases for ….

biobased polymers

2,5-Furan-

dicarboxylic acid

(FDCA)

biomass

glucose/fructose

(PEF)

O

OH

OO

OH

polyethylene

furanoate

Terephthalic acid (PET)

OH

OO

OH

polyethylene

terephthalate

oxidases

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Cupriavidis basilensis HMF14

Dijkman et al. (2013) AMB 97,5177

HMF FDCA

biomass/fructose

predicted

flavoprotein

oxidase

oxidases

R1 F G H R1 A B C D EH′

O

NH

N

R

NH

O

NH

- Original gene & E. coli & plasmids & conditions

- Synthetic genes (!) & E. coli & plasmids & conditions

- Expression in other hosts

- Cupriavidis basilensis HMF14

- Gram-negative, rod-shaped, obligate aerobic

- Isolated for its capacity to degrade HMF

HmfH gene

organism

Getting the enzyme…

Dijkman & Fraaije (2014) AEM 80, 1082

oxidases

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- overexpression in Escherichia coli (100 mg/L culture!)

- soluble and stable monomeric protein

- FAD cofactor as prosthetic group

- Methylovorus sp. Strain MP688: methylotroph

- genome: 2.9 Mb (55 % GC), 2719 ORFs

organism

HmfH homolog (46 % protein seq. ident.)

Getting the enzyme!

Dijkman & Fraaije (2014) AEM 80, 1082

oxidases

- Methylovorus sp. Strain MP688: methylotroph

- genome: 2.9 Mb (55 % GC), 2719 ORFs

organism

HmfH homolog (gene ordered)

HMFOHMFOHMFO

HMF FDCA

Testing the enzyme!

Dijkman & Fraaije (2014) AEM 80, 1082

oxidases

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OH

SH

O

S

OH

OH

OOHOH

OH

O

OH

O

O

SH SH

HMFO also oxidizes thiols!

HMFO takes many compounds

Example substrates

Example reactions

Ewing, Dijkman et al. (2014) Angewandte 53, 13206

O

SH SH

O

SH SH

oxidases

HMF-OH HMF

HMF-acid

FFA FDCA

Crystal structure exploited to tune activity

Pathway of HMF oxidation by HMFO

OOHHO

OOHO

OOHO

HO

OOO

OOO

HO

OOO

HO OH

double mutant was designed

V367R-W466F HFMO

10000x active on FFA!

Crystal structure of HMFO

substrate

oxidases

Dijkman et al. (2015) ACS Catalysis 5, 1833

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Alditol oxidase

Putrescine oxidase

Chitooligosaccharide oxidase

Eugenol oxidase

Hydroxymethylfurfural oxidase

Cytokinin dehydrogenase

Galactonolactone dehydrogenase

O2

O2

O2

O2

CytC

Q

O2

O2

O2

Baron et al. PNAS 2009

oxidases

enzymes discovered @ RUG

Alditol oxidase

• hyperexpression in E. coli

(400 mg/L)

• soluble monomeric protein

• FAD cofactor incorporated

• active with alditols

• Streptomyces coelicolor A3(2): soil bacterium

• notable for production of pharmaceutically useful compounds

• genome: 8.7 Mb (72 % GC), 7825 ORFs

Heuts et al. J. Biol.Chem. 2007

Discovery of alditol oxidase (AldO)

xylitol oxidase homolog identified (76% seq. ident.)

predicted to contain histidyl-FAD

extract!

protein flavin

oxidases

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specific tunnel(s) or, random diffusion to active site?

What determines whether a flavoprotein

can use dioxygen as electron acceptor??

To be or not to be an oxidase

Alditol Oxidase: ideal model enzyme• expression system (high amounts, mutants)

• stable monomeric enzyme, facile purification

• full kinetic mechanism elucidated

• structures: high resolution & complexed structures

1. perform enhanced-statistics MD simulations

2. verify experimentally

Baron et al. PNAS 2009

oxidases

Baron et al.

PNAS 2009

• 1 monomer AldO (all atoms), waters, salt, dioxygens

• 50 ns unbiased calculations (~2 months)

• 500 pathways

Molecular dynamic simulations

oxidases

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Baron et al. PNAS 2009

Molecular dynamic simulations• several defined oxygen tunnels, all in same domain

• tunnels guide oxygen to the same entry point of active site

• entry point is next to reactive C-atom of flavin cofactor

in AldO, last residu to pass

to reach FAD is Ala105…

Ala105Gly mutant is

~2x faster with dioxygen

oxidases

Leferink et al. JBC 2009

IL

A

GP S

A

P

T

oxidase dehydrogenase

IL

A

GP S

A

P

T

oxidase dehydrogenase

oxidase dehydrogenase

Analysis of all characterized AldO-homologs reveals:

glycine at ‘position 105’ is preferred in oxidases

The oxidase-dehydrogenase switch

N C

VAO-type protein sequence

oxidases

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Leferink et al. JBC 2009

IL

A

GP S

A

P

T

oxidase dehydrogenase

IL

A

GP S

A

P

T

oxidase dehydrogenase

oxidase dehydrogenase

Analysis of all characterized AldO-homologs reveals:

glycine at ‘position 105’ is preferred in oxidases

The oxidase-dehydrogenase switch

Is it possible to turn a dehydrogenase

into an oxidase by a single mutation?

Yes we can…

oxidases

L-galactonolactone oxidase

(GALDH A113G mutant)

Ala113Gly renders it an effective oxidase! (400x fold O2 reactivity)

acts as an L-galactonolactone oxidase!!!

O2 H2O2

L-galactono-1,4-lactone L-ascorbic acid

IL

A

GP S

A

P

T

oxidase dehydrogenase

IL

A

GP S

A

P

T

oxidase dehydrogenase

Analysis of all characterized AldO-homologs reveals:

glycine at ‘position 105’ is preferred in oxidases

The oxidase-dehydrogenase switch

Leferink et al. JBC 2009

oxidases

oxidase dehydrogenase

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Alditol oxidase

Putrescine oxidase

Chitooligosaccharide oxidase

Eugenol oxidase

Hydroxymethylfurfural oxidase

Cytokinin dehydrogenase

Galactonolactone dehydrogenase

O2

O2

O2

O2

CytC

Q

O2

O2

Baron et al. PNAS 2009

Galactonolactone dehydrogenase

Galactonolactone oxidase

A113G

Leferink et al. JBC 2009

oxidases

O2

Alditol oxidase

CytC

CytCO2

Alditol oxidase

Putrescine oxidase

Chitooligosaccharide oxidase

Eugenol oxidase

Hydroxymethylfurfural oxidase

Cytokinin dehydrogenase

Galactonolactone dehydrogenase/oxidase

Phenylacetone monooxygenase

Styrene monooxygenase

Trimethylamine monooxygenase

Cyclohexanone monooxygenase

Ethionamide monooxygenase

Steroid monooxygenase

4-hydroxyacetophenone monooxygenase

O2

O2

O2

O2

CytC

NADPH

O2

NADPH

O2

NADPH

O2

NADPH

O2

NADPH

O2

NADPH

O2

NADPH

O2

Q

O2

O2

oxidasesenzymes discovered @ RUG

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Phenylacetone monooxygenase

Styrene monooxygenase

Trimethylamine monooxygenase

Cyclohexanone monooxygenase

Ethionamide monooxygenase

Steroid monooxygenase

4-hydroxyacetophenone monooxygenase

NADPH

O2

NADPH

O2

NADPH

O2

NADPH

O2

NADPH

O2

NADPH

O2

NADPH

O2

NADPHO2

spontaneous

indigo blue

trimethylaminemonooxygenase

monooxygenasesenzymes discovered @ RUG

Alditol oxidase

Putrescine oxidase

Chitooligosaccharide oxidase

Eugenol oxidase

Hydroxymethylfurfural oxidase

Cytokinin dehydrogenase

Galactonolactone dehydrogenase/oxidase

Phenylacetone monooxygenase

Styrene monooxygenase

Trimethylamine monooxygenase

Cyclohexanone monooxygenase

Ethionamide monooxygenase

Steroid monooxygenase

4-hydroxyacetophenone monooxygenase

O2

O2

O2

O2

CytC

NADPH

O2

NADPH

O2

NADPH

O2

NADPH

O2

NADPH

O2

NADPH

O2

NADPH

O2

Q

O2

O2

monooxygenasesenzymes discovered @ RUG

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- overexpression in Escherichia coli

- soluble monomeric protein (62 kDa)

- stable at high temperatures

- tolerant to solvents

- first BVMO crystal structure

- mainly active on aromatic substrates

- Thermobifida fusca: moderate thermophilic soil bacterium

- typical growth temperature: 55°C

- genome: 3.7 Mb (67 % GC), 4587 ORFs

organism

enzyme

motif-based BVMO gene identification

Malito et al., Proc. Natl. Acad. Sci. USA 2004; Fraaije et al., Appl.Microbiol.Biotechnol. 2005; de Gonzalo et al., Tetrahedron: Asymmetry 2005

Phenylacetone monooxygenase (PAMO)

PAMO

NADPH + O2

monooxygenases

- newly accepted substrates by PAMO M446G

- formation of indigo blue

M446 G446

Redesign of substrate binding pocket

Torres Pazmiño et al., Adv. Synth. Catal. 2007

spontaneous

monooxygenases

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BVMOs: useful biocatalysts

BVMOs:

1. Different types of oxidations

2. Structure available

3. Engineering feasible

4. NADPH dependent

NADP+NADPH

monooxygenases

Pazmiño et al. (2008) Angew. Chemie

Self-sufficient BVMOs: PTDH-BVMO fusions

BVMO produced as fusion protein

fused to phosphite dehydrogenase:

1. assures cofactor recycling

2. boosts expression

3. facilitates assay

NADP+NADPH

HPO32- PO4

2-

PTDH

monooxygenases

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Redesign of a monooxygenase

into an oxidase

monooxygenases

O2

substrate

product

oxygenation

H2O2

uncoupling

~10 s-1

~0.01 s-1

NADPH

NADP+ + H2O

Catalytic mechanism of

flavoprotein monooxygenases

monooxygenases

oxidase

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How to destabilize the peroxyflavin?→ generate a NADPH oxidase!

M446

D66C65

Q152

L153

Q152&M446

C65&D66

Q152&L153

libraries

Brondani et al. (2015) JACS

monooxygenases

oxidase

Screening for NADPH oxidase mutants

Dudek et al. (2013) Appl Microbiol Biotechnol.

• Screen for phosphate detection in absence of substrate

O2 + phosphite H2O2 + phosphate

PAMO + PTDH

NADPH NADP+

monooxygenases

oxidase

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How to destabilize the peroxyflavin?→ generate a NADPH oxidase!

M446

D66C65

Q152

L153C65, D66,

Q152, L153,

M446

random

library

Brondani et al. (2015) JACS

one clear hit!

C65D

monooxygenases

oxidase

O2

NADPH

12 s-1/8 s-1

870 M-1s-1/900 M-1s-1

0.014 s-1/25 s-1

300 400 500

C

B

A

0

0.05

0.10

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0,1707 0,1992 0,2337 0,2499 0,2585 0,2683 0,2759 0,2783 0,2673 0,2371 0,195 0,1499 0,1122 0,08504 0,06668 0,05791 0,05395 0,05377 0,05457 0,05709 0,05894 0,06186 0,0649 0,06758 0,07027 0,07334 0,07641 0,0797 0,08479 0,08656 0,08731 0,09024 0,09237 0,09462 0,09629 0,09798 0,09962 0,1002 0,1012 0,1014 0,1013 0,101 0,10070,09987 0,0989 0,09881 0,09543 0,09267 0,08955 0,0859 0,08219 0,07857 0,07508 0,0723 0,06965 0,06833 0,06763 0,06824 0,0674 0,06857 0,07022 0,0723 0,07331 0,07701 0,07913 0,08108 0,08305 0,08511 0,08703 0,08915 0,09166 0,09432 0,09693 0,09948 0,1016 0,1034 0,1037 0,1034 0,1022 0,1004 0,09826 0,09595 0,09504 0,09305 0,09167 0,09058 0,08955 0,08832 0,08681 0,08384 0,08061 0,07665 0,07111 0,065 0,0583 0,05092 0,04415 0,03839 0,03319 0,02885 0,02484 0,02157 0,01948 0,01726 0,0157 0,01436 0,01343 0,01286 0,01238 0,01165 0,01124 0,01112 0,01076 0,01052 0,0104 0,0103 0,009932 0,01034 0,009797 0,009924 0,009539 0,009067 0,009629 0,009535 0,009507 0,009338 0,009366 0,009258 0,009386 0,009071 0,008959 0,009001 0,008983 0,008922 0,008943 0,008815 0,008809 0,00879 0,008201 0,00871 0,008674 0,008581 0,008626 0,008482 0,008468 0,008356 0,008249 0,00821 0,007589 0,00817 0,008168 0,00812 0,00805 0,008052 0,008045 0,007974 0,007953 0,007926 0,007777 0,007741 0,007871 0,007815 0,007762 0,007188 0,007695 0,007656 0,007644 0,007566 0,007552 0,008283 0,007455 0,007907 0,007371 0,0073870,007201 0,007247 0,007198 0,007253 0,007043 0,007941 0,007006 0,006899 0,00816 0,006965 0,006962 0,007049 0,007396 0,007527 0,0069880,006753 0,005635 0,006573 0,006803 0,006619 0,006724 0,006727 0,006678 0,006706 0,006592 0,007664 0,006608 0,005515 0,006576 0,006541 0,006363 0,006179 0,005133 0,006413 0,007522 0,006422 0,006101 0,006509 0,006215 0,00631 0,00779 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0,1707 0,1992 0,2337 0,2499 0,2585 0,2683 0,2759 0,2783 0,2673 0,2371 0,195 0,1499 0,1122 0,08504 0,06668 0,05791 0,05395 0,05377 0,05457 0,05709 0,05894 0,06186 0,0649 0,06758 0,07027 0,07334 0,07641 0,0797 0,08479 0,08656 0,08731 0,09024 0,09237 0,09462 0,09629 0,09798 0,09962 0,1002 0,1012 0,1014 0,1013 0,101 0,10070,09987 0,0989 0,09881 0,09543 0,09267 0,08955 0,0859 0,08219 0,07857 0,07508 0,0723 0,06965 0,06833 0,06763 0,06824 0,0674 0,06857 0,07022 0,0723 0,07331 0,07701 0,07913 0,08108 0,08305 0,08511 0,08703 0,08915 0,09166 0,09432 0,09693 0,09948 0,1016 0,1034 0,1037 0,1034 0,1022 0,1004 0,09826 0,09595 0,09504 0,09305 0,09167 0,09058 0,08955 0,08832 0,08681 0,08384 0,08061 0,07665 0,07111 0,065 0,0583 0,05092 0,04415 0,03839 0,03319 0,02885 0,02484 0,02157 0,01948 0,01726 0,0157 0,01436 0,01343 0,01286 0,01238 0,01165 0,01124 0,01112 0,01076 0,01052 0,0104 0,0103 0,009932 0,01034 0,009797 0,009924 0,009539 0,009067 0,009629 0,009535 0,009507 0,009338 0,009366 0,009258 0,009386 0,009071 0,008959 0,009001 0,008983 0,008922 0,008943 0,008815 0,008809 0,00879 0,008201 0,00871 0,008674 0,008581 0,008626 0,008482 0,008468 0,008356 0,008249 0,00821 0,007589 0,00817 0,008168 0,00812 0,00805 0,008052 0,008045 0,007974 0,007953 0,007926 0,007777 0,007741 0,007871 0,007815 0,007762 0,007188 0,007695 0,007656 0,007644 0,007566 0,007552 0,008283 0,007455 0,007907 0,007371 0,0073870,007201 0,007247 0,007198 0,007253 0,007043 0,007941 0,007006 0,006899 0,00816 0,006965 0,006962 0,007049 0,007396 0,007527 0,0069880,006753 0,005635 0,006573 0,006803 0,006619 0,006724 0,006727 0,006678 0,006706 0,006592 0,007664 0,006608 0,005515 0,006576 0,006541 0,006363 0,006179 0,005133 0,006413 0,007522 0,006422 0,006101 0,006509 0,006215 0,00631 0,00779

300 400 500 600

1000 ms

250 ms

100 ms

50 ms

25 ms

10 ms

1 ms0

0.05

0.10

Ab

so

rba

nce

Wavelength (nm)

Brondani et al. (2015) JACS

C65D PAMO acts as an NADPH oxidase

A

BC

stopped-flow

X-ray structure

NADP+ + H2O2

monooxygenases

oxidase

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Brondani et al. (2015) JACS

C65D PAMO can be used for cofactor regeneration

Substrate Product ADH Temp. (0C)

Time (h)

Conversion (%)

T. brockii 40 24 100a

Evo-1.1.270 30 24 100b

Evo-1.1.270 30 10 50

(ee > 99%)b

Substrate Product ADH Temp. (0C)

Time (h)

Conversion (%)

T. brockii 40 24 100a

Evo-1.1.270 30 24 100b

Evo-1.1.270 30 10 50

(ee > 99%)b

monooxygenases

oxidase

Switching a vitamin-binding protein

into an enzyme

cofactorengineering

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NADPH

NADP+ O2

substrate

product

oxygenationH2O

‘shunt’

H2O2

Catalytic mechanism

cofactorengineering

NADPH

NADP+ O2

substrate

product

oxygenationH2O

‘shunt’

H2O2

Catalytic mechanism

cofactorengineering

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• source: Gallus gallus

• protein: riboflavin-binding protein

• 29 kDa, monomeric

• 10 mg/egg!

• high affinity for flavins (nM range)

• apo preparation protocols

riboflavin-binding protein

Turning riboflavin-binding protein

into an enzyme

cofactorengineering

1 2

alkylated lumiflavin alkylated riboflavin

apo-RfBP + flavin

1 % H2O2, pH 7.5 *

de Gonzalo et al., Chem Comm 2011

flavin c(%)

ee(%)

1 41 27 (S)

2 36 28 (R)

Unnatural flavins

cofactorengineering

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1 2

*apo-RfBP + flavin

1 % H2O2, pH 7.5

flavin c(%)

ee(%)

1 41 27 (S)

2 36 28 (R)de Gonzalo et al., Chem Comm 2011

cofactorengineering

natural flavin → unnatural flavin

oxidations

reductions

light sensing

e- transport

hydrolysis

halogenation

light production

magnetosensing

H2O2-driven oxidations???

cofactorengineering

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EncM

enterocin

Teufel et al. (2013) Nature 503:552-556 Payne et al. (2015) Nature 522:497-501White et al. (2015) Nature 522:502-506

N

N

O

N H

NH

O

R

new natural flavins!

(de)carboxylationsFavorskii rearrangement

cofactorengineering

Conclusions

Flavin-containing enzymes: versatile biocatalysts

to be continued…

• Flavoenzymes are highly tuneable

Insight into enzyme mechanism can be extremely helpful

to introduce new activities:

dehydrogenase → oxidase (single mutation!)

monooxygenase → oxidase (single mutation!)

monooxygenase-dehydrogenase fusion

flavin cofactors can be engineered

flavoenzymes

18-10-2017

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Marco FraaijeMolecular Enzymology

University of Groningen

The Netherlands

m.w.fraaije@rug.nl

Andrea Mattevi

University of Pavia

Willem van Berkel

Wageningen University

Riccardo Baron

UC San Diego

Acknowledgements

funding collaborators materials

Fraaije group - September 2016www.dinosaurusmuseum.nl

www.gecco-biotech.com