Citation for published version:Zaitsev, V, Johnsen, U, Reher, M, Ortjohann, M, Taylor, G, Danson, M, Schönheit, P & Crennell, S 2018,'Insights into the Substrate Specificity of Archaeal Entner-Doudoroff Aldolases: The Structures of Picrophilustorridus 2-Keto-3-deoxygluconate Aldolase and Sulfolobus solfataricus 2-Keto-3-deoxy-6-phosphogluconateAldolase in Complex with 2-Keto-3-deoxy-6-phosphogluconate', Biochemistry, vol. 57, no. 26, pp. 3797-3806.https://doi.org/10.1021/acs.biochem.8b00535DOI:10.1021/acs.biochem.8b00535
Publication date:2018
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Insights into substrate specificity of archaeal Entner-
Doudoroff aldolases: the structures of Picrophilus torridus 2-keto-3-deoxygluconate aldolase, and Sulfolobus
solfataricus 2-keto-3-deoxy(6-phospho)-gluconate aldolase in complex with 2-keto-3-deoxy-6-phosphogluconate.
Journal: Biochemistry
Manuscript ID bi-2018-00535u.R1
Manuscript Type: Article
Date Submitted by the Author: n/a
Complete List of Authors: Zaitsev, Viatcheslav; University of St Andrews, Biomolecular Sciences
Johnsen, Ulrike; Christian-Albrechts Universität Kiel, Reher, Matthias; Christian-Albrechts Universität Kiel Ortjohann, Marius; Christian-Albrechts Universität Kiel Taylor, Garry; University of St Andrews, Danson, Michael; University of Bath, Biology & Biochemistry Schönheit, Peter; Christian-Albrechts Universität Kiel, Institut für Allgemeine Mikrobiologie Crennell, Susan; University of Bath, Biology & Biochemistry
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Insights into substrate specificity of archaeal Entner-Doudoroff aldolases: the structures of 1
Picrophilus torridus 2-keto-3-deoxygluconate aldolase, and Sulfolobus solfataricus 2-keto-3-2
deoxy(6-phospho)-gluconate aldolase in complex with 2-keto-3-deoxy-6-phosphogluconate. 3
Viatcheslav Zaitseva, Ulrike Johnsenb, Matthias Reherb, Marius Ortjohannb, Garry L.Taylora, Michael J. 4
Dansonc, Peter Schönheitb and Susan J. Crennellc# 5
6
Biomolecular Sciences, University of St Andrews, St Andrews, Fife, UKa; Institut für Allgemeine 7
Mikrobiologie, Christian-Albrechts-Universität, Kiel, Germanyb; Department of Biology & Biochemistry, 8
University of Bath, Bath, UKc. 9
10
V.Z. and U.J. contributed equally to this work 11
12
# Corresponding author: Susan Crennell, [email protected] 13
14
Second contributing author: Peter Schönheit [email protected] 15
16
Running title: Picrophilus and Sulfolobus KDG-Aldolase specificity 17
18
Abbreviations. 19
20
KDG: 2-keto-3-deoxygluconate 21
KDPG: 2-keto-3-deoxy-6-phosphogluconate 22
KDG-aldolase: 2-keto-3-deoxygluconate aldolase 23
KDGK: KDG kinase 24
KD(P)G-aldolase: 2-keto-3-deoxygluconate/2-keto-3-deoxy-6-phosphogluconate aldolase 25
Pt-KDG-aldolase: Picrophilus torridus KDG-aldolase 26
RMSD: Root Mean Square Deviation 27
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Ss-KD(P)G-aldolase: Sulfolobus solfataricus KD(P)G-aldolase 28
29
Table of Contents graphic (Figure7) 30
31
EC number: 4.1.2.51 32
33
Keywords: Picrophilus torridus, 2-keto-3-deoxygluconate aldolase, crystal structure, enzyme 34
specificity, Sulfolobus solfataricus, 2-keto-3-deoxy-phospho-gluconate aldolase, 35
archaeal Entner-Doudoroff pathways 36
37
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Abstract 38
39
The thermoacidophilic archaea Picrophilus torridus and Sulfolobus solfataricus catabolise glucose via 40
a non-phosphorylative Entner-Doudoroff pathway and a branched Entner-Doudoroff pathway, 41
respectively. Key enzymes for these Entner-Doudoroff pathways are the aldolases, 2-keto-3-42
deoxygluconate aldolase (KDG-aldolase) and 2-keto-3-deoxy-6-phosphogluconate aldolase (KD(P)G-43
aldolase). KDG-aldolase from P. torridus (Pt-KDG-aldolase) is highly specific for the non-44
phosphorylated substrate, 2-keto-3-deoxygluconate (KDG), whereas KD(P)G-aldolase from 45
S. solfataricus (Ss-KD(P)G-aldolase) is an enzyme catalyzing the cleavage of both KDG and 2-keto-3-46
deoxy-6-phosphogluconate (KDPG), with a preference for KDPG. The structural basis for the high 47
specificity of Pt-KDG-aldolase for KDG as compared to the more promiscuous Ss-KD(P)G-aldolase 48
has not been analysed before. In the current paper we report the elucidation of the structure of Ss-49
KD(P)G-aldolase in complex with KDPG at 2.35Å and that of KDG-aldolase from P. torridus at 2.50 Å 50
resolution. By superimposition of the active sites of the two enzymes, and subsequent site-directed 51
mutagenesis studies, a network of four amino acids, namely Arg106, Tyr132, Arg237 and Ser241, was 52
identified in Ss-KD(P)G-aldolase that interact with the negatively-charged phosphate group of KDPG, 53
thereby raising the affinity of the enzyme for KDPG. This KDPG-binding network is absent in Pt-KDG-54
aldolase, which explains the low catalytic efficiency of KDPG cleavage. 55
56
57
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Introduction 58
The discovery of thermophilic micro-organisms has stimulated a wealth of fundamental and applied 59
research into their enzymes, both for the light they may shed on the molecular basis of protein stability 60
and for the potential applications of enzymes with high thermal stability. Most extreme thermophiles 61
are members of the Archaea, the third domain of life, and studies on their metabolic pathways have 62
revealed enzymes with unusual catalytic activities and specificities. 63
The Archaea degrade glucose and glucose polymers to pyruvate, not via the classical 64
Embden-Meyerhof pathway nor the Entner-Doudoroff pathway reported for Bacteria and Eukarya, but 65
via modifications of these pathways, thereby implicating novel enzymes and enzyme families with 66
unusual catalytic and regulatory features.1,2,3 Modified Embden-Meyerhof pathways have been 67
described mainly in anaerobic hyperthermophilic archaea whereas aerobic archaea, including the 68
thermoacidophiles Picrophilus torridus and Sulfolobus solfataricus, degrade glucose via modified 69
versions of the Entner-Doudoroff pathway. For P. torridus, growing optimally at 60°C and pH 0.9, a 70
non-phosphorylative Entner-Doudoroff pathway was reported.4,5 Accordingly, glucose is converted to 71
2-keto-3-deoxygluconate (KDG) via glucose dehydrogenase and gluconate dehydratase. KDG is then 72
cleaved by a KDG-aldolase to pyruvate and glyceraldehyde, of which the latter is converted to a 73
second molecule of pyruvate via glyceraldehyde dehydrogenase, 2-phosphoglycerate-forming 74
glycerate kinase, enolase and pyruvate kinase (Fig. 1). 75
76
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77
78 Fig 1 Modified Entner-Doudoroff (ED) pathways in P. torridus and S. solfataricus. KDG, 2-keto-3-79
deoxygluconate; GA, glyceraldehyde; 2-PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; KDPG, 80
2-keto-3-deoxy-6-phosphogluconate; GAP, glyceraldehyde-3-phosphate; 3-PG, 3-phosphoglycerate; 81
GDH, glucose dehydrogenase; GAD, gluconate dehydratase; KDGA, KDG aldolase; GADH, 82
glyceraldehyde dehydrogenase; GLK, glycerate kinase (2-PG forming); ENO, enolase; PYK, pyruvate 83
kinase; KD(P)GA, KDG/KDPG aldolase; KDGK, KDG kinase; GAOR, glyceraldehyde oxidoreductase; 84
GAPN, non-phosphorylative GAP dehydrogenase; PGM, phosphoglycerate mutase. The C4 epimeric 85
substrate galactose and its respective derivatives of GDH, GAD, KDGK, KDGA and KD(P)GA are not 86
shown. 87
Non-phosphorylative ED pathway
P. torridus
Branched ED pathway S. solfataricus
Glucose
Gluconate
KDG
GDH
Pyruvate
GA Pyruvate
Glycerate
2-PG
PEP
NAD(P)H
GAD
KDGA
GADH
GLK
ENO
PYK
Glucose
Gluconate
KDG
GA Pyruvate
Glycerate
Pyruvate
2-PG
PEP
KDPG
GAP Pyruvate
3-PG
KD(P)GA
KDGK
NAD(P)H
ATP
ADP ADP
ATP
XH2
NAD(P)H
ATP
NAD(P)H GDH
GAD
GAOR
GLK
ENO
PYK
PGM
GAPN
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88
On the other hand, S. solfataricus, growing at 80°C and pH 3.5, possesses a branched Entner-89
Doudoroff pathway.6,7 In this pathway, glucose is converted to KDG via glucose dehydrogenase and 90
gluconate dehydratase as in P. torridus. Further catabolism of KDG involves two branches; in the first, 91
KDG is cleaved to pyruvate and glyceraldehyde, which is further converted to pyruvate via 92
glyceraldehyde oxidoreductase, glycerate kinase, enolase and pyruvate kinase. In the second branch, 93
KDG is phosphorylated by KDG kinase to generate 2-keto-3-deoxy-6-phosphogluconate (KDPG), 94
which is cleaved to pyruvate and glyceraldehyde-3-phosphate by a KD(P)G-aldolase that catalyzes 95
the cleavage of both KDG and KDPG. Further conversion of glyceraldehyde-3-phosphate to pyruvate 96
involves a non-phosphorylative glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate 97
mutase, enolase, and pyruvate kinase (Fig. 1). 98
Both the non-phosphorylative Entner-Doudoroff pathway of P. torridus and the branched 99
Entner-Doudoroff pathway of S. solfataricus have been described to be promiscuous for the 100
catabolism of both D-glucose and D-galactose .4,5,7,8 101
The substrate specificities of the Picrophilus and Sulfolobus aldolases are key determinants of 102
their respective metabolic pathways. KDG-aldolase (E.C. 4.1.2.51) from P. torridus is highly specific 103
for the substrate KDG, showing up to 2000-fold higher catalytic efficiency compared to KDPG.4 In 104
contrast, KD(P)G-aldolases are enzymes catalyzing the cleavage of both KDG and KDPG, with a 105
preference for KDPG. KD(P)G-aldolases have been characterized from the Sulfolobus species, 106
S. solfataricus, S. acidocaldarius and S. tokodaii, and from Thermoproteus tenax .6,7,9 Both types of 107
aldolases, KDG-aldolase and KD(P)G-aldolase, are Schiff-base-forming class I aldolases that belong 108
to the dihydrodipicolinate synthase-like family of the class I aldolase superfamily, where they form 109
distinct clusters in accordance with their different substrate specificities .4 It should be noted that the 110
archaeal KDG-aldolase and KD(P)G-aldolase differ from the KDPG aldolases of the classical Entner-111
Doudoroff pathway of bacteria and of the semi-phosphorylative Entner-Doudoroff pathway of the 112
haloarchaeon Haloferax volcanii,10 which belong to the bacterial type class I KDPG aldolases/4-113
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hydroxy-2-oxoglutarate aldolase family .11 114
While the differences in substrate specificities of KDG-aldolase and KD(P)G-aldolase are in 115
accordance with their in vivo operation in the non-phosphorylative and branched Entner-Doudoroff 116
pathways, respectively, the structural basis for the high specificity of Pt-KDG-aldolase for KDG as 117
compared to the more promiscuous KD(P)G-aldolase from Sulfolobus has not been analysed so far. 118
However, the structures of KD(P)G-aldolases from S. solfataricus (Ss-KD(P)G-aldolase), 119
S. acidocaldarius and T. tenax have been determined. KDPG was modelled into the active site of the 120
S. acidocaldarius KD(P)G-aldolase 9 and of T. tenax KD(P)G-aldolase,12 and from these structural 121
models two arginines and one tyrosine have been proposed to contact the phosphate group of KDPG. 122
To analyse the different substrate specificities of the aldolases further, a direct structural 123
comparison of the KDG-aldolase with KD(P)G-aldolase is necessary. Therefore, in the current paper, 124
we report the elucidation of the structure of KDG-aldolase from P. torridus, specific for the non-125
phosphorylated substrate KDG, and also the structure of the KD(P)G-aldolase from S. solfataricus in 126
complex with KDPG. By subsequent superimposition of the active sites of the two enzymes, we 127
identify amino acids that contact the phosphate group of KDPG in Ss-KD(P)G-aldolase and the 128
corresponding residues in Pt-KDG-aldolase. Subsequent predictions of the structural basis of the 129
respective substrate specificities are then tested by site-directed mutagenesis. 130
131
132
133
134
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Materials and Methods 135
Site-directed mutagenesis 136
Variants of KDG aldolase from P. torridus (Pt-KDG-aldolase) and of KD(P)G aldolase from S. 137
solfataricus (Ss-KD(P)G-aldolase) were generated using the QuikChange Lightning Site-directed 138
Mutagenesis Kit (Agilent Technologies, Waldbronn, Germany) or the Q5 Site-directed Mutagenesis Kit 139
(New England Biolabs, Frankfurt, Germany), according to the manufacturers’ instructions. The 140
plasmids pET3a-SSO_3197 8 and pET19b-Pto1279a 4 were used as templates. 141
142
Expression and purification of enzymes 143
Pt-KDG-aldolase and Ss-KD(P)G-aldolase, and their respective variants, were expressed in E. coli 144
(DE3) Rosetta pLysS cells grown at 37°C for 4 h in Lysogeny Broth (LB) medium. Expressions were 145
induced by the addition of isopropyl β-D-1-thiogalactopyranoside. Recombinant Pt-KDG-aldolase and 146
variants of the enzyme were purified by heat treatment at 55°C for 30 min, followed by affinity 147
chromatography on Ni-NTA Agarose according to Reher et al. (2010). Eluted KDG-aldolase was then 148
applied to a Superdex 200 HiLoad 16/60 column (GE Healthcare, Freiburg, Germany) that was 149
equilibrated with 50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl. Protein was eluted with an 150
isocratic flow. At this stage, Pt-KDG-aldolase and variants were essentially pure as judged by sodium 151
dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). 152
Cell pellets of recombinant Ss-KD(P)G-aldolase and site-directed variants were suspended in 153
50 mM Tris-HCl, pH 8.6, and passed three times through a French press cell; cell extracts were 154
generated by centrifugation at 39,000 g. Purification was performed by heat treatment at 75°C for 30 155
min followed by centrifugation at 39,000 g. The resulting supernatant was applied to a Q Sepharose 156
HP HiLoad 16/10 column (GE Healthcare) that was equilibrated in 50 mM Tris-HCl, pH 8.6. The 157
protein was eluted with an increasing gradient from 0 to 2 M NaCl. Fractions containing highest 158
KD(P)G-aldolase activity were applied to a Superdex 200 HiLoad 16/60 column. Elution of protein was 159
performed in 50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl. After this procedure, the protein was 160
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essentially pure as judged by SDS-PAGE. 161
162
Determination of enzyme activity 163
Aldol-cleavage activities of Pt-KDG-aldolase and Ss-KD(P)G-aldolase were measured by following the 164
production of pyruvate from KDG or KDPG. Assays were carried out at 60°C in 50 mM sodium 165
phosphate buffer, pH 6.2, containing 0.3 mM NADH, 3 U lactate dehydrogenase, and KDPG (Sigma-166
Aldrich, Schnelldorf, Germany; product 79156) or KDG (Sigma-Aldrich, product 12271); the reduction 167
of pyruvate to lactate by NADH, catalysed by lactate dehydrogenase, was monitored 168
spectrophotometrically at 340nm.4 169
170
Crystallisation, X-ray data collection, processing and model refinement 171
The genes encoding Pt-KDG-aldolase and Ss-KD(P)G-aldolase were cloned and expressed, and the 172
recombinant enzymes purified and characterised, as described above. For crystallization, Pt-KDG-173
aldolase was concentrated to 4.2 mg/ml and, following the identification of initial crystallisation 174
conditions (Crystal Screen Cryo HT, Hampton Research), optimal crystals were obtained by the 175
hanging-drop, vapour-diffusion method, using drops of protein and well solution (0.085 M sodium 176
HEPES buffer pH 7.5, 8.5% (v/v) 2-propanol, 17% (w/v) PEG 4000, 15% (v/v) anhydrous glycerol) in 177
equal ratio. Ss-KD(P)G-aldolase, concentrated to 6mg/ml, was also crystallised by the hanging-drop, 178
vapour-diffusion method using drops containing 1.5 µl of protein mixed with 1.5 µl of well solution 179
(0.1M sodium HEPES buffer pH 6.0, 8% (v/v) 2-propanol, 11% (w/v) PEG4000)13. The Ss-KD(P)G-180
aldolase crystals were soaked in well solution containing 15mM KDPG (Sigma) and 20% v/v glycerol 181
(as cryoprotectant) for 5 min, followed by flash-cooling in liquid nitrogen. 182
X-ray data were collected from Pt-KDG-aldolase crystals at 100K using a Rigaku MicroMax -183
007 HF with Saturn 944+ CCD detector. Diffraction spots were visible to at least 2.4Å; however, during 184
data processing the resolution was truncated to 2.5Å to optimise data completeness. X-ray data were 185
processed and scaled using HKL-2000.14 The molecular replacement pipeline BALBES15 was used to 186
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find initial phases using the structure of Bacillus anthracis 4-hydroxy-tetrahydrodipicolinate synthase 187
DapA-2 (1XKY,16 with which Pt-KDG-aldolase shares 32% identity) as the search model. Iterative 188
cycles of model building in Coot17 and refinement in Phenix 18 were required to build a final model. 189
X-ray data were collected from the SsKD(P)G-aldolase complex crystals at 100K on the I04 190
beamline at the Diamond Light source, using a Dectris Pilatus 6M-F detector. 720° of data were 191
collected using radiation of wavelength 0.9795Å and processed using DIALS,19 then scaled using 192
AIMLESS,20 truncated to 2.35Å to achieve completeness, with high CC(1/2) and sufficient signal 193
(mean I/σ(I)). BALBES15 was again used to solve the structure, using the unliganded Ss-KD(P)G-194
aldolase structure (1W37) as the starting model, and cycles of rebuilding in Coot and refinement in 195
Phenix. The KDPG ligand structure was built and the Schiff base conformation and CIF file produced 196
using JLigand.21 197
Except where indicated, PyMOL22 was used to produce the figures in this paper. 198
199
Size-exclusion chromatography 200
For determination of the native relative molecular masses (Mr) of Pt-KDG-aldolase, Ss-KD(P)G-201
aldolase and their respective variants, a Superdex 200 HiLoad 16/60 column was calibrated with high-202
molecular-weight (HMW) and low-molecular-weight (LMW) kits (GE Healthcare, Germany) as 203
specified by the manufacturer. The subunit Mr values were calculated from the protein sequences and 204
were seen to be consistent with estimates from Coomassie-stained SDS-PAGE. 205
206
Accession number: The Pt-KDG-aldolase structure has been deposited in the PDB and given the 207
accession number 4UXD, DOI: 10.2210/pdb4uxd/pdb. The Ss-KD(P)G-aldolase structure in complex 208
with KDPG has been given the accession number 6G3Z. 209
210 211
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Results and Discussion 212
213 Overall Pt-KDG-aldolase structure: 214
The structure of Pt-KDG-aldolase has been determined to 2.5 Å, deposited in the PDB and given the 215
code 4UXD. Data processing and structure refinement data are given in Table 1. The asymmetric unit 216
contains one Pt-KDG-aldolase tetramer, 19 glycerol (cryoprotectant) molecules, three molecules of 217
propan-2-ol, five of ethane-1,2-diol, two PEG derivatives (PEG, PGE), and 340 water molecules. The 218
structure is of high quality, with a good Molprobity score (1.90, 97th percentile), although 6 residues 219
were classified as Ramachandran plot outliers, these being residue 105 in chains A, C and D, residue 220
77 in chains A and C, and residue 207 in chain C. All these residues are marginal outliers, not seen in 221
all chains; however, the most consistent outlier is Tyr105 and its unusual conformation can be seen to 222
be caused by its position on the dimer interface, projecting into a pocket in the other monomer (Fig. 2). 223
Fig 2: The structure of Pt-KDG-aldolase in cartoon representation, with each monomer in a different 224
colour. The Ramachandran-outliers Tyr 105 are shown in stick form, and can be seen to protrude into 225
a neighbouring molecule as part of the larger dimer interface, the perpendicular tetramer interface 226
being less extensive. 227
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Table 1: Data processing and model refinement statistics. Numbers in parentheses refer to the 228
highest resolution shell in each data set (2.54-2.50Å for Pt-KDG-aldolase, 2.43-2.35Å for Ss-KD(P)G-229
aldolase). 230
231
Data processing Pt-KDG-aldolase Ss-KD(P)G-aldolase
Unit Cell Dimensions (Å) 77.9, 100.2, 154.6 106.9, 106.9, 245.6
Space Group P22121 P6522
Resolution (Å) 28.58-2.50 (2.54-2.50) 92.55-2.35 (2.43-2.35)
Completeness (%) 86.6 (83.4) 100.0 (100.0)
Rmerge 0.107 (0.333) 0.145 (2.51)
Rpim (all I+ & I-) 0.017(0.284)
Redundancy 3.8 (2.8) 75.8 (79.1)
I/σI 13.2 (2.7) 22.2 (2.7)
Number of observed reflections 877347 2692983 (269399)
Number of unique reflections 36955 (1730) 35510 (3406)
Model refinement
Number of working set reflections 34773 35153
Number of test set reflections 1884 1686
Final Rcryst 0.175 0.187
Final Rfree 0.260 0.239
Number of non-H protein atoms 8979 4838
Number of non-H solvent atoms 340 79
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RMSD bonds (Å) 0.008 0.009
RMSD angles (°) 1.18 1.00
Ramachandran plot most favoured (%) 95.8 96.74
Ramachandran plot allowed (%) 3.7 3.26
232
The asymmetric unit contains 4 chains, each of which folds into a classic (βα)8 TIM-barrel 233
structure, and their structural similarity is shown by the root mean square deviation (RMSD) values of 234
any pair of superimposed independently-refined monomers being no greater than 0.3Å. Analysis of Pt-235
KDG-aldolase by PISA23 indicates that these monomers form a tetramer as a dimer of dimers, with the 236
average dimer interface having an area of 1610Å2 and a solvation energy for folding (∆Gf) of -20 237
kcal/mol, while that between dimers in the tetramer is only 680Å2 and -5.6kcal/mol. 238
Comparison with other structures in the PDB using PISA clearly identifies Pt-KDG-aldolase as 239
a member of the dihydrodipicolinate synthetase family, having marked structural similarity with the 240
many structures in 'Aldolase Class 1' (CATH superfamily 3.20.20.70). Several structures of KD(P)G-241
aldolase have been published, including those from T. tenax (RMSD to Pt-KDG-aldolase of 1.9Å over 242
259 Cα, sequence identity 21.7%), Sulfolobus acidocaldarius (2.0Å over 274 Cα, sequence identity 243
27.9%), and S. solfataricus (1.8Å over 259 Cα, sequence identity 24.8%). Despite the low sequence 244
identity between these KD(P)G-aldolases, and the somewhat larger RMSD values, indicating less 245
similarity with Pt-KDG-aldolase than the approximately 1.0Å RMSD calculated when these structures 246
are compared to each other, the structural similarity is clear (see Fig. 3 for alignment of Ss-KD(P)GA 247
and Pt-KDG-aldolase). All these structures have also been solved in the presence of pyruvate, 248
glyceraldehyde or KDG, and no significant movement of the framework is seen on binding. 249
250
Overall structure of Ss-KD(P)G-aldolase in complex with KDPG. 251
To investigate the interactions of KDPG with a KD(P)G-aldolase, the structure of Ss-KD(P)G-aldolase 252
in complex with KDPG was determined to 2.35Å; data processing and model refinement statistics are 253
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given in Table 1. The overall structure is very similar to both the unliganded (1W37) and the KDG-254
bound (1W3N) structures (average RMSD over all 293 Cα: 0.3Å). Following refinement of the 255
unliganded coordinates, there was clear difference density for a ligand bound in the active site, 256
extending beyond the KDG seen in the 1W3N structure. (Fig 4B). However the density was too broad 257
to fit a single phosphate position, and therefore the final model has KDPG in a dual conformation, with 258
the two positions occupied approximately equally (0.52:0.48), and has been deposited in the PDB with 259
the code 6G3Z. 260
261
262
263
Fig 3. Cartoon representation of Ss-KD(P)G-aldolase (grey) aligned to Pt-KDG-aldolase (green) to 264
show the overall structural similarity. The active site is marked by a stick representation of the Lysine 265
(155 and 159 respectively) which forms the Schiff base in the catalytic reaction, and which is bound to 266
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KDPG (yellow) in the Ss-KD(P)G-aldolase complex structure. The three N-terminal amino acids of the 267
Pt-KDG-aldolase are removed for clarity. 268
269
Active site 270
The structure of Ss-KD(P)G-aldolase in complex with KDG has been described previously13. As noted 271
by Theodossis et al. for this structure, in comparison with the unliganded enzyme (1W37), the majority 272
of the Ss-KD(P)G-aldolase active site is unchanged on binding KDG, although there are minor 273
movements of aromatic side chains (Phe 39 and Tyr 130) that are mirrored in the KDPG-bound 274
structure. As well as these hydrophobic interactions, the same hydrogen-bonding interactions between 275
the KDG moiety and the enzyme are seen in the two structures (the C1 acid group with the main chain 276
of both Thr43 and Thr44 and the Thr44 side chain, O4 with Tyr130 and O6 with Tyr132 ) (Fig 4B). 277
However, the KDPG-bound structure shows that the more distal part of the active site involved in 278
phosphate interactions is somewhat less rigid; on binding KDPG, the Cα positions of Arg106, and in 279
some monomers Glu159 (which forms salt bridges with Arg106 and Arg273; Fig 5B), are the only non-280
terminal Cα amino acids to move more than 1Å from their positions in the earlier structures. The 281
movement of Arg106 can be explained through both phosphate conformations of KDPG making strong 282
interactions (within hydrogen-bonding distance) with Arg106 from a neighbouring monomer (Fig 5B). 283
The other interactions with the phosphates are from residues within the original monomer, conformer A 284
interacting with Ser241 and Tyr132, while conformer B interacts with Arg237. 285
Through modeling studies, the interactions with Arg237, Tyr132 and Arg106 have been 286
predicted previously in Sa-KD(P)G-aldolase (234, 105, 131)9 and Tt-KD(P)G-aldolase (255, 150, 287
124)12, being conserved across other KD(P)G aldolases, while the Ser 241, which is not conserved, 288
had not been identified in those studies. 289
In all the KD(P)G-aldolase structures, the active site containing the lysine that will form the 290
Schiff base is in a conserved position at the top of the β-barrel. As was also seen in S. acidocaldarius 291
KD(P)G-aldolase when crystallised at pH 7.5, in the Pt-KDG-aldolase structure the lysine points away 292
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from the pyruvate binding pocket into the centre of the barrel (Fig. 3); however, in S. acidocaldarius 293
KD(P)G-aldolase it was shown that no other amino acids moved significantly between this 294
arrangement and the catalytically-competent configuration seen at pH 6.3.9 The heightened mobility of 295
this residue in Pt-KDG-aldolase is shown by the large temperature factor of the lysine Nζ (50.2Å2) 296
relative to the average of side chain atoms (36.8Å2), and the reduced electron density at 1.5 σ(Figure 297
4A). 298
299
Fig 4. Following superimposition of the structures, the active sites of (A) the apo structure of Pt-KDG-300
aldolase (GOL are glycerol molecules) and (B) Ss-KD(P)G-aldolase with KDPG (yellow bonds) bound 301
are shown in the same orientation with the 2FoFc electron density map for each structure, contoured 302
at 1.5σ, restricted to that around KDPG in B for clarity. B also contains the structure of Ss-KD(P)G-303
aldolase with KDG bound (grey) superimposed to show the relative stasis of all but those amino acids 304
that interact with the phosphates. The main chain cartoon containing Arg106 from a neighbouring 305
monomer is omitted as it crosses the middle of the image. Hydrogen bonds are shown as dotted lines. 306
307
Comparison of the inner part of the binding-pocket seen in the Ss-KD(P)G-aldolase-KDPG complex 308
structure ( Fig. 4B)20 with the Pt-KDG-aldolase active site (Fig. 4A) shows that many of the KDG 309
interactions would be conserved in the Picrophilus enzyme; these comprise interactions of the 310
pyruvate carboxylate group with Ss-KD(P)GA Thr43 (Pt-KDG-aldolase Ser44), Thr44(45), 311
Tyr130(131), and the glyceraldehyde with Thr157(Ser161) and, via water, with Gly179(184), Thr44(45) 312
and Ala198(Gly203). The suitability of the site to bind polar molecules in Pt-KDG-aldolase is shown by 313
the presence of glycerol (cryoprotectant) molecules, making many of the same interactions as KDG in 314
Ss-KD(P)G-aldolase (Fig. 4A). The interaction of the O6 of KDG with Ss-KD(P)G-aldolase Tyr132 315
would not be replicated in Pt-KDG-aldolase as the corresponding residue is Ile133. Both enzymes 316
have shown promiscuity in catalyzing the cleavage of both KDG and KDGal, 4,7 so it is likely that the 317
mechanism of action, as observed in Ss-KD(P)G-aldolase,7 is conserved in Pt-KDG-aldolase due to 318
A B
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the similarities in the inner part of the binding pocket. 319
320
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321
322
Fig 5: Analysis of interactions (shown in magenta dotted lines) made by KDPG with the active sites of 323
Pt-KDG-aldolase (modeled on the complex between KDPG and Ss-KD(P)G-aldolase) (A,C), and 324
those seen in Ss-KD(P)G-aldolase (B,D). (A) Stick representations of amino acids from Pt-KDG-325
aldolase (green) that could interact with KDPG (magenta), with those amino acids from neighbouring 326
subunits in different shades of green. (B) A similar representation of amino acids in the Ss-KD(P)G-327
aldolase active site, with those from a neighbouring monomer in a different shade of blue. (C) 328
Electrostatic surface around the catalytic site of Pt-KDG-aldolase, with positively-charged areas in 329
blue and negative in red. (D) Electrostatic surface around the catalytic site of Ss-KD(P)G-aldolase, 330
coloured similarly. 331
332
C
A
D
B
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333
Unlike the other KD(P)G aldolases of known structure, the Pt-KDG-aldolase has a distinct 334
preference for KDG over KDPG as a substrate, the catalytic efficiency with KDG being 2,000 times 335
that with KDPG.4 To investigate this specificity in the outer part of the binding pocket, the structure of 336
Pt-KDG-aldolase was compared to that of Ss-KD(P)G-aldolase in complex with KDPG (Fig. 5B). None 337
of the Ss-KD(P)G-aldolase phosphate-interacting residues is conserved in Pt-KDG-aldolase, the site 338
of Arg237 being occupied by glycine (241), and Ser241 by phenylalanine (245), neither of which will 339
form constructive interactions with a phosphate moiety. The main chain of Arg106 of Ss-KD(P)G-340
aldolase aligns with Lys107 in Pt-KDG-aldolase, also a positive amino acid, but the latter forms an 341
interaction within its own subunit, and is not available for substrate interaction (Fig. 5A). The entrance 342
to the active site surface is considerably more hydrophobic in Pt-KDG-aldolase (Fig 5C), although 343
some positive charge is provided by Arg166 from the subunit on the other side (Fig. 5A), which could 344
make longer-distance interactions with one phosphate conformation. However the other phosphate 345
conformation would be unfavourable due to clashes, particularly with Phe 245, and the hydrophobic 346
nature of these outer reaches of the Pt-KDG-aldolase catalytic pocket is increased further by Ile133 347
(Pt) instead of Tyr132 (Ss), and Phe136 (Pt) replacing Ala135 (Ss) (Fig. 5C,D). These differences are 348
predicted to explain the lower affinity of Pt-KDG-aldolase for phosphorylated substrates. 349
350
Site-directed mutagenesis 351
To investigate these active-site observations further, a series of residues was swapped by site-352
directed mutagenesis and the resulting kinetic analyses of the variant enzymes are summarised in 353
Table 2; kinetic analyses from selected variants are shown graphically in Supplementary Figures S1 354
and S2. Initial studies concentrated on three of the residues in Ss-KD(P)G-aldolase that interact with 355
the phosphate group of the substrate KDPG, namely Arg237, Arg106 and Tyr132 . Thus Ss-KD(P)G-356
aldolase R237G raised the KM for KDPG approximately 5-fold with no significant change in the Vmax. 357
There were no significant changes in the kinetic parameters with KDG as substrate. The reverse 358
change in Pt-KDG-aldolase, G241R, increased the kcat/KM for KDPG 9-fold but, surprisingly, this came 359
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from an increase in kcat rather than an expected decrease in the KM; as predicted, no change was 360
observed in the kinetic parameters with KDG as substrate. 361
362
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Table 2: Kinetic parameters determined for mutated Ss and Pt aldolases. 363
364
S. solfataricus KD(P)G Aldolase 365
KDPG as substrate Vmax (U/mg) KM (mM) kcat (s-1) kcat/KM (s-1M-1)
Native enzyme 168.4 ± 17.3 0.69 ± 0.09 97.8 ± 10.1 1.42 (± 0.24) x 105
R237G 174.4 ± 9.3 3.28 ± 0.28 101.1 ± 5.4 0.31 (± 0.03) x 105
R106I 157.6 ± 9.2 0.98 ± 0.15 91.4 ± 5.3 0.93 (± 0.15) x 105
Y132I 332.1 ± 43.8 0.68 ± 0.03 192.6 ± 25.3 2.83 (± 0.39) x 105
R106I/Y132I 141.6 ± 13.4 1.57 ± 0.33 82.1 ± 7.8 0.52 (± 0.12) x 105
S241F 200.4 ± 6.6 0.89 ± 0.08 116.2 ± 3.8 1.31 (± 0.13) x 105
S241F/L242P 114 ± 4.2 0.58 ± 0.07 66.1 ± 2.4 1.14 (±0.14) x 105
R106I/Y132I/S241F 85.3 ± 3.9 2.45 ± 0.20 49.5 ± 2.3 0.20 (± 0.02) x 105
366
367
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P. torridus KDG Aldolase 368
Vmax (U/mg) KM (mM) kcat (s-1) kcat/KM (s-1M-1)
KDPG as substrate
Native enzyme 0.785 ± 0.07 6.4 ± 1.5 0.398 ± 0.035 62.14 ± 15.6
G241R 6.77 ± 0.56 6.27 ± 0.9 3.43 ± 0.283 546.98± 90.6
KDG as substrate
Native enzyme 62.4 ± 11.6 1.12 ± 0.3 31.6 ± 5.88 2.82 (± 0.91) x 104
G241R 63.4 ± 9.4 1.06 ± 0.08 32.12 ± 4.78 3.03 (± 0.51) x 104
369
In each active site of Ss-KD(P)G-aldolase, Arg106 from a neighbouring subunit interacts with 370
the phosphate group of KDPG, and accordingly the mutation R106I raised the KM for KDPG, although 371
the effect was much smaller (1.4-fold) than that for R237G. The residue in the equivalent position in 372
the Picrophilus enzyme is Lys107; attempts were therefore made to reposition this Lys to one similar 373
to Arg106 in Ss-KD(P)G-aldolase. Using the sequence alignment as a guide (Fig. 6), a Pro was 374
inserted in Pt-KDG-aldolase before Lys107, and Phe245 was changed to Ser245 as the bulky Phe 375
appeared to be blocking the binding of the phosphate of KDPG; unfortunately, the enzyme lost over 376
70% of the wild-type enzyme activity, and size exclusion chromatography indicated that the enzyme 377
was dissociating. The F245S mutant without the insertion of the Pro also had no defined oligomeric 378
structure. 379
380
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381 Fig 6: Structure-based sequence alignment (calculated with Expresso24) and displayed with Espript25 382
of Pt-KDG-aldolase (KDGA_PICTO), Thermoplasma acidophilum KDG-aldolase (DAPAL_THEAC), S. 383
acidocaldarius KD(P)G-aldolase (KDGA_SULAC), Ss-KD(P)G-aldolase (KDGA_SULSF) and T. tenax 384
KD(P)G-aldolase (KDGA_THETE), with the sequences labeled with their Uniprot codes and the 385
secondary structure depicted above the sequences. Identical amino acids are in white text on a red 386
background; similar amino acids are in red text. The four amino acids labeled in cyan are those shown 387
to interact with the phosphate group of KDPG in Ss-KD(P)G-aldolase, R106, Y132, R237 and S241 388
(Ss numbering). Those in orange are thought to make phosphate binding less likely I133, G241, F245 389
(Pt numbering). 390
391
392
Surprisingly, mutation of Y132 to Ile did not affect the KM of Ss-KD(P)G-aldolase for KDPG, but 393
increased kcat 1.9 fold; the reason for this is perhaps that in the absence of Y132, which interacts with 394
conformation A, the phosphate preferentially occupies the other position in the active site and the 395
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replacement with the smaller Ile allows a more open active site. However, the mutation of Y132 in 396
combination with R106I, affecting both phosphate binding sites, did increase the KM two-fold over 397
R106I alone. Mutation of I133Y in Pt-KDG-aldolase, to mimic the Ss-KD(P)G-aldolase Y132, was 398
attempted but caused dissociation of the oligomeric structure with considerable loss of catalytic 399
activity. 400
Ser241 forms a hydrogen bond with the phosphate of KDPG in Ss-KD(P)G-aldolase, whereas 401
this position is occupied by a Phe in Pt-KDG-aldolase, which will not form hydrogen bonds and 402
appears to obstruct the access of the phosphorylated substrate to the active site, particularly in one of 403
the two KDPG conformations. However, the mutation S241F in Ss-KD(P)G-aldolase had only a small 404
effect on the catalysis of KDPG. This may be because only one of the two phosphate binding 405
conformations is affected; moreover catalysis with KDG as substrate was similarly unaffected. Also, in 406
Pt-KDG-aldolase the Phe-containing loop takes up a different conformation from that containing S241 407
in Ss-KD(P)G-aldolase, possibly due to the Pro that lies underneath the Phe; consequently, the double 408
mutant, S241F/L242P, aimed to allow the Phe to take up the Picrophilus-like conformation, but again 409
little effect on the binding of KDPG was observed. However, the influence of Ser241 on the binding of 410
KDPG was best demonstrated in the triple mutant R106I/Y132I/S241F, where a 4-fold increase in KM 411
was observed; that is, doubling the increase seen in the double mutant R106I/Y132I. Thus, Ser241 412
contributes to KDPG binding as part of a network with R106 and Y132 and together with R237, 413
constitutes the fourth amino acid of the network. 414
415
Concluding remarks 416
In this communication we report the 3D structure of Pt-KDG-aldolase, which is highly specific for KDG, 417
and the structure of Ss-KD(P)G-aldolase in complex with KDPG, where the phosphate was 418
unexpectedly shown to bind in two conformations. By structural comparison of Pt-KDG-aldolase and 419
KDPG-cleaving Ss-KD(P)G-aldolase, and by site-directed mutagenesis studies, a network of four 420
amino acids composed of Arg 106, Tyr 132, Arg 237 and Ser241 was identified that contacts the 421
phosphate of KDPG in Ss-KD(P)G-aldolase (Figure 7). This KDPG-binding network was absent in Pt-422
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KDG-aldolase, explaining the low catalytic efficiency of this aldolase for KDPG. The respective 423
substrate specificities of the two aldolases for KDG and KDPG determine the two different routes of 424
glucose degradation via non-phosphorylative or branched Entner-Doudoroff pathway in these two 425
thermophilic archaea (Fig 1). The operation of the non-phosphorylative Entner-Doudoroff pathway in P. 426
torridus is further supported by the absence of a KDG-kinase gene in this organism. 427
428 429
430 Fig 7. Interactions (magenta dotted lines with lengths in Å) of a four amino acid-network (Arg106, 431
Tyr132, Arg237 and Ser241) predicted from structural data and shown through site-directed 432
mutagenesis to be important for binding the phosphate group of KDPG in Ss-KD(P)G-aldolase. Ss-433
KD(P)G-aldolase amino acids from one monomer in cyan, from a neighbour in dark blue, and KDPG in 434
yellow). 435
436 The presence or the absence of the four amino acid network in sequences of homologous 437
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aldolases enables the prediction of substrate specificities of these aldolases and their functional 438
involvement in archaeal Entner-Doudoroff pathways. The presence of the network in aldolases from 439
Sulfolobus acidocaldarius and Thermoproteus tenax (see structure based alignment in Fig 6.) is in 440
accordance with the kinetic properties showing high preference for KDPG over KDG; both organisms 441
have been reported to degrade glucose via a branched Entner-Doudoroff pathway .4,6 Furthermore, 442
the absence of the network in the aldolase from Thermoplasma acidophilum is in accordance with the 443
high specificity for KDG over KDPG and with the operation of the non-phosphorylative Entner-444
Doudoroff pathway in this organism.4 445
446
447 Author contributions: MR and MO produced the proteins for crystallisation. VZ crystallised Pt-KDG-448
aldolase and with GLT collected X-ray data on Pt-KDG-aldolase crystals; SJC solved and refined the 449
Pt-KDG-aldolase crystal structure and also crystallised and solved the structure of the Ss-KD(P)G-450
aldolase complex with KDPG. MO and UJ performed the mutagenesis experiments and MJD, PS, 451
and UJ analysed the enzyme kinetic data . PS initiated the project and SJC, PS, MJD and UJ have 452
been involved in writing the paper. 453
454
Acknowledgements 455
Initial work of site-directed mutagenesis studies was performed by Jasmin Gille as part of her bachelor 456
thesis (Christian-Albrechts-Universität Kiel). Refinement of the Pt-KDG-aldolase structure was carried 457
out as part of the work submitted by Alexandros Priftis for an MRes in Protein Structure and Function 458
at the University of Bath. The authors would like to thank Diamond Light Source for beamtime 459
(proposal mx17212-8), and Gyles Cozier, William Bradshaw, Shalini Iyer and the staff of beamline i04 460
for assistance with data collection. This research received no specific grant from any funding agency 461
in the public, commercial or not-for-profit sectors. None of the authors have any real or perceived 462
conflicts of interest. 463
464
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Supporting Information. Kinetic analyses of wild-type and mutant aldolases from Sulfolobus (Figure 465
S1) and Picrophilus (Figure S2). 466
467
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References 468
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implementations and evaluation of a new integration package. Acta Crystallogr. D74, 85-97. 524
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Figure 2: The structure of Pt-KDG-aldolase in cartoon representation, with each monomer in a different colour. The Ramachandran-outliers Tyr 105 are shown in stick form, and can be seen to protrude into a
neighbouring molecule as part of the larger dimer interface, the perpendicular tetramer interface being less
extensive.
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Fig 3. Cartoon representation of Ss-KD(P)G-aldolase (grey) aligned to Pt-KDG-aldolase (green) to show the overall structural similarity. The active site is marked by a stick representation of the Lysine (155 and 159 respectively) which forms the Schiff base in the catalytic reaction, and which is bound to KDPG (yellow) in the Ss-KD(P)G-aldolase complex structure. The three N-terminal amino acids of the Pt-KDG-aldolase are
removed for clarity.
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Figure 4A
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Fig 4. Following superimposition of the structures, the active sites of (A) the apo structure of Pt-KDG-aldolase (GOL are glycerol molecules) and (B) Ss-KD(P)G-aldolase with KDPG (yellow bonds) bound are shown in the same orientation with the 2FoFc electron density map for each structure, contoured at 1.5σ
restricted to that around KDPG in B for clarity. B also contains the structure of Ss-KD(P)G-aldolase with KDG bound (grey) superimposed to show the relative stasis of all but those amino acids that interact with the phosphates. The main chain cartoon containing Arg106 from a neighbouring monomer is omitted as it
crosses the middle of the image. Hydrogen bonds are shown as dotted lines.
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Figure 5A
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Figure 5B
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Figure 5C
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Fig 5: Analysis of interactions (shown in magenta dotted lines) made by KDPG with the active sites of Pt-KDG-aldolase (modeled on the complex between KDPG and Ss-KD(P)G-aldolase) (A,C), and those seen
in Ss-KD(P)G-aldolase (B,D). (A) Stick representations of amino acids from Pt-KDG-aldolase (green) that
could interact with KDPG (magenta), with those amino acids from neighbouring subunits in different shades of green. (B) A similar representation of amino acids in the Ss-KD(P)G-aldolase active site, with those from a neighbouring monomer in a different shade of blue. (C) Electrostatic surface around the catalytic site of Pt-KDG-aldolase, with positively-charged areas in blue and negative in red. (D) Electrostatic surface around
the catalytic site of Ss-KD(P)G-aldolase, coloured similarly.
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jjjjk iiiiiiiiiiiii jjk lll iiiiiiiiiiiii
jjk iiiiiiiiiiiii jjjk iiiiiiiiiiii jjjjjk ii
iiiiiiii jjjjjk iiiiiiii jjjk llliiiiii jjk llll iiiiiiii iiii
iiiiii iiiiiiiii iiiiii iiii .
jjjjk iiiiiiiiiiiii jjjk lll iiiiiiiiiii
j jjk iiiiiiiiiiii jjjk iiiiiiiiiiiii i jjjjk iiii ii .. ..
ii jjjjjk iiiiiiiiii jjjk llliiiii jjk llll iiiiiiiiiiii iiii
iiiii iiiiiiiiii iiiiiiii iiiii iiiiiiiiiiiiiiiiiiiii . .
jjjjk iiiiiiiiiiiii jjjk lll lll iiiiiiiiiiii
j jjk iiiiiiiiiilll jjjjk iiiiiiiiiiiii i jjjjk iiii ii .. ..
iiiii jjjjjk iiiiiiiiii jjjk llliiiiii jjk llll iiiiiiiiiiii iiii
iiiii iiiiiiiiii iiiiiiii iiiii iiiiiiiiiiiiiiiiiiii . .
jjjjk jk jk iiiiiiiiiiiii jjjk lll iiiiiiiiiiiii
i j jjk iiiiiiiiiiiii jjjk iiiiiiiiiiiii i jjjjk iiii ii .. ..
iiiii jjjjjk iiiiiiiiii jjjk llliiiiii jk lll iiiiiiiiiiii iiii
iiiii iiiiiiiiiiii iiiiiiii iiiii iiiiiiiiii iiiii . .
X X X X X X X KDGA_PICTO 1 c c cc c c c cc c c DF DEEEF A DF A A DF DEF DF DEEEF A A DF A A A I P IT D F G TG P E V M P G ID A I L V L S V T R L V EMYK....................G C LDAH N YN TNIL KY EGIN Y PM F YF LK KDF KF R
DAPAL_THEAC 1 c c cc c c c cc c c KG KdddG B KG B B KG KdG KG KdddG B B KG B B B I P IT D F G TG P E V M P G ID A I NL GV L S L S K L V DMYK....................G T MGQN E YR TEIL D ADF G PM F MF TD KKF GF R
KDGA_SULAC 1 c c cc c c c cc c c KG KdddG B KG B B KG KdG KG KdddG B B KG B B B I P IT D F G TG P E I I P G V A A NL GI I T L S K L L D..M....................E S FDKQ K NVD LKTH K LEK A VN G AL KD RQN NA Y
KDGA_SULSF 1 c c cc c c c cc c c KG KdddG B KG B B KG KdG KG KdddG B B KG B B B I P IT D F G TG P E I I P ID A NL GI L T L S K L V D.MP....................E T FTKDNR KEKLKIH E IRK K VN G SL PE LEN KA Y
KDGA_THETE 1 c c cc c c c cc c c JH JIIIH C JH C C JH JIH JH JIIIH C C JH C C C I P IT D F G TG P E V V G LD L V NI GV V T L S K A ..MSHPLIPFRANFLRAPRVLSME A TFR.G R PE FANH K TSK V VA G AL LQ MELTDA TS
X X X X X X X X KDGA_PICTO 66 c c c cc cccc DF A A DEF DF DEF DEEEF A A DF DEF DF A A DEF A A DEEEF A DF A V L K YY YN P SK I M GS SI EV M FS D I AV P KL I HYY I N L I T I N KP .. AG S N NE M IG EA LMP I. NQEA Y KE LS..S DMD L I QF .NK. DPE
DAPAL_THEAC 66 c c c cc cccc KG B B KdG KG KdG KdddG B B KG KdG KG B B KdG B B KdddG B KG B V L K YY YN P SK I GS S E V YT D I V K E M R F V N LFI LS I H K. EVYAG S TQ S E S E IG KVR LMPT I. PD DW Y H ST ISAAS D.. I Q .GSW SES
KDGA_SULAC 64 c c c cc c cccc KG B B KdG KG KdG KdddG B B KG KdG KG B B KdG B B KdddG B KG B V L K YY YN P TH L I GS NL DVM V FS E I V P RL E L KYY I S LYI AT I V .K .. FQ L N E N MD LG SSHS FP P KF A EE AR..I SHS Y A GYD. PPS
KDGA_SULSF 65 c c c cc c cccc KG B B KdG KG KdG KdddG B B KG KdG KG B B KdG B B KdddG B KG B V L K YY YN P T I I G NL DAI A S D I IA P RM E L KYF L S VYL AT I V N.K .. FQ GL D R L K FD VG SYA YP S KH V KT CE..V PHP Y T GKD. DAK
KDGA_THETE 83 c c c cc c cccc JH C C JIH JH JIH JIIIH C C JH JIH JH C C JIH C C JIIIH C JH C V L K YY YN P R V I S NA EAI A Y A VA P RL E I KYF L VFL A V AA .R .. VQ A L D A AESRG EA SLP FP S RQ A RD CS..AVSIP Y A VGRD. DAR
X X X X X X X X KDGA_PICTO 144 c cc c c c c c cDEF A DEEEEEEF A A DEEEEF DEF DF A A A DEF A DF A DF A G KD G L G V N A VK L VK V SS I M VFQ QD LF SL A T F D IV L E T N KSEFSS AD RGF EMLSLSDDDFA D T ELG S G CGT S. G R YHEYK.NN.R M
DAPAL_THEAC 146 c cc c c c c c cKdG B KddddddG B B KddddG KdG KG B B B KdG B KG B KG B G KD G L G V N ALI L VK I SS M IFQ QD LF SL A S I L LE EK TREFSN GD RFFSRIIRHKNEKFD D L SIG S G CGL IS.PY TN YHEFSAGN. K
KDGA_SULAC 143 c cc c c c c c cKdG B KddddddG B B KddddG KdG KG B B B KdG B KG B KG B G KD G L G V N AILK L VK I TN L L VYN S IY SL L T F E IV LD S .....P QD AHS EYKLNLPG.VK NT Y LS. D V ASF IP V KQRDLIK.QGK D
KDGA_SULSF 144 c cc c c c c c cKdG B KddddddG B B KddddG KdG KG B B B KdG B KG B KG B G KD G L G V N AVAK I V T I L VYS SD I A L S Y E V I ID E ....GCFT IEN IHT DYKRLNPN.ML M ATV STG D N AAG LP VT T KKLAM.ERK E
KDGA_THETE 162 c cc c c c c c cJIH C JIIIIIIH C C JIIIIH JIH JH C C C JIH C JH C JH C G KD G L G V N AAAK L IR V TN L L VYN SD VF S L Y E LA I IE E ....GC ES AHT AYKRYLPQ.AR S A FAVR D V ASSA LP L G RDAVA.AGD R
X X X X X KDGA_PICTO 226 c c c c c c DEF A DF A A DF DEEEF A A Y R P E KI V LM L N P MY V L EKND IP KK GKYQFPNAY EY.FYKK NING.GY ........R GI I.......................
DAPAL_THEAC 229 c c c c c c KdG B KG B B KG KdddG B B Y R P E KIQ V LM I NG M I R IDE NP YA NEATFPAGY .YAFYKM IKG.GY A VEPTTDQKKK DQ LTKIPKKQ................
KDGA_SULAC 220 c c c c c c KdG B KG B B KG KdddG B B Y R P E RLQ I LA L QG P IF L L E.L NR DI RKYGSISAI VL.VNEF YDVGYP ........P TD EALSLKREIEPLKRKIQELVH...
KDGA_SULSF 223 c c c c c c KdG B KG B B KG KdddG B B Y R P E KLQ VI QG P IF L L F.LHDE EASRIFGSLSSN VL.TKYF YDLGYP ........P DD EERQLIKKVEGIRAKLVELKILKE
KDGA_THETE 241 c c c c c c JIH C JH C C JH JIIIH C C Y R P E LQ L IV A QG G VY L RS F.L DE ES RHIGYAAAV EL.VEIF YEAGEP ........P DP EKAWLRAAVAKAKSQLRL......
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Fig 7. Interactions (magenta dotted lines with lengths in Å) of a four amino acid-network (Arg106, Tyr132, Arg237 and Ser241) predicted from structural data and shown through site-directed mutagenesis to be
important for binding the phosphate group of KDPG in Ss-KD(P)G-aldolase. Ss-KD(P)G-aldolase amino acids from one monomer in cyan, from a neighbour in dark blue, and KDPG in yellow).
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Table of Contents graphic (Figure 7 rotated)
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