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, S.J.Crennell@bath.ac.uk 13

14

Second contributing author: Peter Schönheit peter.schoenheit@ifam.uni-kiel.de 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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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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