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1

Substrate recognition mechanism and substrate-dependent conformational 1

changes of an ROK family glucokinase from Streptomyces griseus 2

Running title: Structure of an ROK family glucokinase from S. griseus. 3

4

Ken-ichi Miyazono,1 Nobumitsu Tabei,1 Sho Morita,2 Yasuo Ohnishi,2 Sueharu Horinouchi,2,3 and Masaru 5

Tanokura1* 6

1. Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The 7

University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 8

2. Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of 9

Tokyo, 1-1-1 Yayoi Bunkyo-ku, Tokyo 113-8657, Japan 10

3. Deceased on 12th July 2009 11

12

*Corresponding author 13

Correspondence to: Dr. Masaru Tanokura 14

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences 15

The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 16

Phone: +81-3-5841-5165 17

Fax: +81-3-5841-8023 18

E-mail: [email protected]

Formatted: Numbering: Continuous

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.06173-11 JB Accepts, published online ahead of print on 18 November 2011

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Abstract 20

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Carbon catabolite repression (CCR) is a widespread phenomenon in many bacteria that is defined as 22

the repression of catabolic enzyme activities for an unfavorable carbon source by the presence of a preferable 23

carbon source. In Streptomyces, secondary metabolite production is often negatively affected by the carbon 24

source, indicating the involvement of CCR in secondary metabolism. Although the CCR mechanism in 25

Streptomyces is still unclear, glucokinase is presumably a central player in CCR. SgGlkA, a glucokinase from 26

S. griseus, belongs to the ROK family glucokinases, which have two consensus sequence motifs (1 and 2). 27

Here we report the crystal structures of apo-SgGlkA, SgGlkA in complex with glucose, and SgGlkA in 28

complex with glucose and AMPPNP, which are the first structures of an ROK family glucokinase. SgGlkA is 29

divided into a small α/β domain and a large α+β domain, and forms a dimer of dimer tetrameric configuration. 30

SgGlkA binds a β-anomer of glucose between the two domains, and His157 in the consensus sequence 1 plays 31

an important role in the glucose-binding mechanism and anomer specificity of SgGlkA. In the structures of 32

SgGlkA, His157 forms an HC3-type zinc-finger motif with three cysteine residues in the consensus sequence 33

2 to bind a zinc ion and forms two hydrogen bonds with the C1 and C2 hydroxyls of glucose. When the three 34

structures are compared, the structure of SgGlkA is found to be modified by the binding of substrates. The 35

substrate-dependent conformational changes of SgGlkA may be related to the CCR mechanism in 36

Streptomyces. 37

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Introduction 39

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The Gram-positive, soil-dwelling, filamentous bacterial genus Streptomyces is characterized by its 41

complex morphological differentiation (spore germination - vegetative mycelium - aerial mycelium - spores) 42

and by its ability to produce a wide variety of secondary metabolites. Because of the commercial importance 43

of the secondary metabolites for applications such as antibiotics, immunosuppressants, insecticides, and 44

anti-tumor agents, there is great interest in Streptomyces as an industrial microorganism (7, 8, 11). Production 45

of secondary metabolites in Streptomyces is often genetically coupled with morphological differentiation and 46

is frequently negatively affected by the carbon source (19, 44, 50). This regulatory phenomenon, broadly 47

termed carbon catabolite repression (CCR), is achieved by a variety of regulatory mechanisms (6, 13). For the 48

effective production of secondary metabolites by Streptomyces, it is important to understand the CCR 49

mechanism of this genus. 50

CCR is a widespread phenomenon in many bacteria that can be defined, in a narrow sense, as the 51

repression of enzyme activities for the catabolism of an unfavorable carbon source by the presence of a 52

preferable catabolite in the growth medium. CCR is important for many bacteria to win the struggle for 53

survival in the natural environment, because the selection of a carbon source is closely related to the growth 54

rate of the organisms. CCR mechanisms in gram-negative bacteria such as Escherichia coli and 55

low-G+C-content gram-positive bacteria such as Bacillus subtilis have been well characterized (13). CCR in E. 56

coli is mediated by the prevention of transcriptional activation of catabolic genes in the presence of glucose, 57

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while CCR in B. subtilis is mediated by negative regulation by a repressor protein in the presence of glucose 58

(13). Although the molecular mechanisms of CCR are different, the phosphoenolpyruvate:carbohydrate 59

phosphotransferase system (PTS) plays an important role for the CCR mechanisms of both organisms. On the 60

other hand, the CCR mechanism in high-G+C-content gram-positive bacteria, including Streptomyces, is little 61

understood. Streptomyces seems to have no glucose PTS (40, 52). In Streptomyces coelicolor A3(2), glucose is 62

incorporated into the cell by the major facilitator superfamily sugar permease GlcP (57). So far, glucokinase seems 63

to be a central player in CCR; glucokinase has been suggested to have a regulatory function as well as a metabolic 64

function (3, 4, 27, 31), although at least two articles have contradicted these conclusions (21, 42). Interestingly, van 65

Wezel et al. reported that glucokinase was activated posttranslationally in a glucose transport-dependent manner in 66

S. coelicolor A3(2) (56). 67

Glucokinase catalyzes the phosphorylation of glucose to yield glucose 6-phosphate using ATP. 68

Microbial glucokinases are classified into three groups (Groups I - III) (30). Group I consists of ATP- and 69

ADP-dependent glucokinases from archaea and eukaryotes (PFAM accession number PF04587) (43, 45). The 70

ADP-dependent glucokinases are involved in a modified Embden-Meyerhof pathway in archaea. The 71

structural analyses of group I glucokinases (22, 23, 54) show that the structures of group I glucokinases are 72

similar to those of ATP-dependent kinases such as E. coli ribokinase and human adenosine kinase (33, 47). 73

Group II glucokinases are ATP-dependent glucokinases that do not contain the ROK (repressors, open reading 74

frames, and kinases) sequence motif (PFAM accession number PF02685). This group includes the 75

well-characterized E. coli glucokinase (EcGlk) (30, 36). The structure of EcGlk shows structural similarity to 76

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Saccharomyces cerevisiae hexokinase B and human brain hexokinase I (1, 2), and the structure is distinct 77

from those of group I glucokinases. Group III consists of glucokinases belonging primarily to Gram-positive 78

bacteria and archaea, and contains the ROK sequence motif (consensus sequences 1 and 2) (PFAM accession 79

number PF00480) (35, 51). Consensus sequences 1 and 2 consist of 28 amino acid residues with a conserved 80

ExGH motif and of 14 residues with a conserved Zn-binding motif (CXCGXXGCXE), respectively (Fig. 1). 81

In addition, the ATP-binding motif, active site loop, and catalytic Asp are also conserved in the ROK family 82

proteins (9). A global regulator of sugar metabolism in Gram-negative bacteria, Mlc, is one of the ROK family 83

proteins. The structure of E. coli Mlc, which is the first-described structure of the ROK family protein, shows 84

that Mlc is divided into the DNA-binding domain, the EIIB-binding domain, and the oligomerization domain 85

(39, 46). Among them, the EIIB-binding and oligomerization domains show amino acid sequence similarities 86

to microbial group III glucokinases. In the structure of Mlc, the His residue in the ExGH motif and three 87

cysteine residues in the Zn-binding motif form an HC3-type zinc-finger to bind a zinc ion (39, 46). Although 88

cysteine residues in the Zn-binding motif are indispensable for the enzymatic activity of group III 89

glucokinases (35), the reason why these cysteine residues are important for the catalytic mechanism of 90

glucokinase remains unclear, because no structures of these group glucokinases are currently known. 91

Glucokinase from Streptomyces griseus (SgGlkA) is included in this microbial glucokinase family. 92

Here we report the crystal structures in apo, glucose-bound, and glucose- and AMPPNP-bound states 93

of SgGlkA at 3.25 Å, 1.84 Å, and 1.55 Å resolutions, respectively. These are the first structures of a member 94

of the Group III microbial glucokinases. The structures of SgGlkA show that SgGlkA forms a dimer of 95

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dimer-tetrameric configuration and the histidine residue in the zinc-finger motif conserved in the ROK family 96

is involved in the glucose recognition mechanism. The comparison of the three structures of SgGlkA shows 97

that the structure of SgGlkA is modified by the binding of glucose and AMPPNP. This structural modification 98

by the bindings of substrates may be related to the glucose-dependent catabolite repression mechanism in 99

Streptomyces; it may provide SgGlkA with a regulatory function as a kind of glucose sensor. 100

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Materials and Methods 102

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Protein expression, purification, crystallization, and data collection 104

The protein expression and purification of SgGlkA were performed as described previously (37). All 105

the crystallization experiments were performed by the sitting-drop vapor-diffusion method at 20ºC. 106

Crystallization of SgGlkA in complex with glucose was performed as described previously. For the 107

crystallization of apo-SgGlkA, the purified protein was dialyzed against 10 mM Tris-HCl, pH 8.0, and 108

concentrated to 20 mg/ml. Crystals of apo-SgGlkA were obtained in reservoir solution containing 0.1 M 109

Tris-HCl, pH 7.5, 16% PEG3350, and 0.2 M ammonium sulfate. For the crystallization of 110

SgGlkA-glucose-AMPPNP, the purified protein was dialyzed against 10 mM Tris-HCl, pH 8.0, and 50 mM 111

D-glucose and was supplemented with 5 mM MgCl2 and 5 mM AMPPNP. The protein solution was 112

concentrated to 13.3 mg/ml for crystallization. Crystals of SgGlkA-glucose-AMPPNP were obtained in a 113

reservoir solution containing 0.1 M Tris-HCl, pH 7.2, 0.75 M sodium citrate, and 0.2 M NaCl. 114

X-ray diffraction data sets of apo-SgGlkA, SgGlkA-glucose, and SgGlkA-glucose-AMPPNP were 115

collected at beamline BL-17A of the Photon Factory (Tsukuba, Japan). The X-ray diffraction data set of 116

SgGlkA-glucose was collected and processed as described previously (37). The crystals of apo-SgGlkA and 117

SgGlkA-glucose-AMPPNP were soaked in the corresponding reservoir solutions supplemented with 20% 118

ethylene glycol before being flash-cooled at 95 K in a nitrogen stream. The best crystals of apo-SgGlkA and 119

SgGlkA-glucose-AMPPNP diffracted X-rays to 3.25 Å and 1.55 Å resolutions, respectively. The diffraction 120

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data were indexed, integrated, and scaled with XDS (24). The crystal of apo-SgGlkA belonged to the primitive 121

monoclinic space group P21, with unit cell parameters of a = 98.69 Å, b = 173.70 Å, c = 124.03 Å, and β = 122

106.69o. The crystal of SgGlkA-glucose-AMPPNP belonged to the orthorhombic space group I222, with unit 123

cell parameters of a = 69.58 Å, b = 87.73 Å, and c = 125.39 Å. The data collection statistics are summarized 124

in Table 1. 125

126

Structure determination 127

The structure of SgGlkA-glucose was determined by the molecular replacement method. The initial 128

model of SgGlkA-glucose was determined using the program MOLREP (55) with the coordinates of putative 129

N-acetylmannosamine kinase (PDB ID 2AA4) as a search model. After the molecular replacement, the 130

structure of SgGlkA-glucose was refined and manually rebuilt using the programs Refmac5 (38) and 131

XtalView (34). The structures of apo-SgGlkA and SgGlkA-AMPPNP-glucose were determined by the 132

molecular replacement method using the program MOLREP with the coordinates of SgGlkA-glucose. Model 133

building and refinement were performed using the same method described above. The geometries of the final 134

structures were evaluated using the program Rampage (29). The refinement statistics of the structures are 135

summarized in Table 1. 136

137

Computational analysis 138

The structures of SgGlkA were analyzed using a set of computer programs: Dalilite for the 139

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superposition of molecules (17), PISA for the analysis of protein interfaces, surfaces, and assemblies (26), 140

ALAdeGAP for the amino acid sequence alignment (16), ESpript for the preparation of alignment figures (12), 141

and Pymol for the depiction of structures (10). 142

143

Data bank accession code 144

Coordinates and structure factors for the apo-SgGlkA, SgGlkA-glucose, and 145

SgGlkA-glucose-AMPPNP have been deposited in the RCSB Protein Data Bank under the accession codes 146

3VGK, 3VGM, and 3VGL, respectively. 147

148

Oligomeric state analysis by gel-filtration chromatography 149

The purified SgGlkA was loaded onto a Superdex 200 HR 10/30 (GE Healthcare) column and eluted 150

under two buffer conditions: 20 mM Tris-HCl, pH 8.0, 200 mM NaCl, and 50 mM D-glucose, or 20 mM 151

Tris-HCl, pH 8.0, and 200 mM NaCl. To estimate the multimerization state of SgGlkA, the following standard 152

proteins were used: aldolase (Mr = 158,000), canalbumin (Mr = 75,000), ovalbumin (Mr = 43,000), and 153

carbonic anhydrase (Mr = 29,000). The experiments were performed at 4ºC. 154

155

Enzyme assays 156

The coupling assay with glucose 6-phosphate dehydrogenase as a coupling enzyme was used in the 157

kinetic experiment of SgGlkA (48). The formation of glucose 6-phosphate was coupled to the reduction of 158

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NADP by glucose 6-phosphate dehydrogenase. The reaction (total volume 300 μl) was carried out at 30ºC in 159

50 mM Tris-HCl, pH 8.0, containing 100 mM KCl, 1 mM NADP, ATP (4.3 or 0.86 mM), MgCl2 (1 mM in 160

excess of the ATP concentrations), glucose (0.125-5 mM), SgGlkA (0.055 mg/ml), and glucose 6-phosphate 161

dehydrogenase (2.24 units/ml). The reduction of NADPH was measured spectrophotometrically by 162

monitoring the absorbance at 340 nm. To determine kinetic parameters, the data were fit to the 163

Michaelis-Menten equation. 164

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Results and discussion 166

167

Overall structure of SgGlkA 168

The structures of apo-SgGlkA, SgGlkA in complex with glucose (SgGlkA-glucose), and SgGlkA in 169

complex with glucose and AMPPNP (SgGlkA-glucose-AMPPNP) were determined by the molecular 170

replacement method at resolutions of 3.25 Å, 1.84 Å, and 1.55 Å, respectively, with good stereochemistry. 171

Although we used the same protein sample for the crystallizations of SgGlkA, we could not get a better 172

resolution for the apo-structure. This may have been at least partly due to the potential flexibility of 173

apo-SgGlkA in the crystalline state. But the exact reason for the poor resolution remained unclear, because the 174

qualities of crystals are easily affected by the crystallization conditions. The final models of apo-SgGlkA, 175

SgGlkA-glucose, and SgGlkA-glucose-AMPPNP contained eight, one, and one SgGlkA molecules in the 176

asymmetric units, respectively, and refined to R/Rfree values of 21.2/26.5%, 16.8/18.5%, and 17.9/20.2%, 177

respectively. Although we did not add zinc ion during the purification and crystallization steps of SgGlkA, 178

each protomer of SgGlkA contains one zinc ion around the Zn-binding motif of consensus sequence 2. In 179

addition, two sulfate ions, one potassium ion, and one sodium ion are also observed in the structures of 180

apo-SgGlkA, SgGlkA-glucose, and SgGlkA-glucose-AMPPNP, respectively. These ions are contained in the 181

crystallization conditions of apo-SgGlkA, SgGlkA-glucose, and SgGlkA-glucose-AMPPNP as ammonium 182

sulfate, potassium/sodium tartrate, and sodium citrate, respectively. In the Ramachandran plot, 94.2% of the 183

apo-SgGlkA residues, 99.0% of the SgGlkA-glucose residues, and 99.4% of the SgGlkA-glucose-AMPPNP 184

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residues were in the favored region, and the rest were in the allowed region. The data collection and 185

refinement statistics are summarized in Table 1. 186

The protomer structures of SgGlkA-glucose and SgGlkA-glucose-AMPPNP are shown in Figure 2. 187

SgGlkA-glucose consists of 13 β-strands, 10 α-helices, and 4 310 (η)-helices and is divided into two domains 188

(an N- and C- terminal small α/β domain and a central large α+β domain). The small domain (resides 1–120 189

and 297–313) consists of a five-stranded mixed β-sheet (the strand order is β3-β2-β1-β4-β8, with strand β2 190

antiparallel to the others), a three-stranded antiparallel small β-sheet (the strand order is β5-β6-β7), and four 191

α-helices (α1, α2, α3, and α10) surrounding the β-sheets. The helices α1 and α2 face the mixed β-sheet at 192

one side, and helices α3 and α10 face the other side. The large domain (residues 121–296) consists of a 193

five-stranded mixed β sheet (the strand order is β11-β10-β9-β12-β13, with strand β10 antiparallel to the 194

others) and six α helices (α4, α5, α6, α7, α8, and α9) facing one side of the mixed β sheet. The other side of 195

the mixed β sheet faces the small domain. 196

197

Multimerization of SgGlkA 198

It is known that bacterial glucokinases form monomeric, dimeric, and tetrameric structures. The 199

majority of bacterial glucokinases described so far are dimeric proteins (14). In contrast, it is known that 200

glucokinases from Streptomyces coelicolor and Streptomyces peucetius var. caesius, which show 85% amino 201

acid sequence identities with SgGlkA, form tetrameric assemblies (20). Although S. coelicolor glucokinase is 202

stable in its tetrameric form, S. peucetius var. caesius glucokinase forms a homotetramer only in the presence 203

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of 100 mM glucose. S. peucetius var. caesius glucokinase easily dissociates into a dimeric form in the absence 204

of glucose (20). In this study, the structures of apo-SgGlkA, SgGlkA-glucose, and SgGlkA-glucose-AMPPNP 205

form homotetrameric structures in those crystals, although the crystallization conditions and the space groups 206

of the crystals are different (Fig. 3AB). The oligomerization state of SgGlkA in solution was also analyzed by 207

size exclusion chromatography (Fig. 3C). The molecular weights of SgGlkA in the absence or presence of 208

glucose are estimated to be 120,000 and 140,000, respectively. This result indicates that SgGlkA also shows a 209

homotetrameric assembly in solution (the molecular weight of the SgGlkA protomer is 32,336) and the 210

homotetrameric structure of SgGlkA is slightly affected by the presence of glucose. The structural difference 211

of apo-SgGlkA and SgGlkA-glucose will be discussed later. 212

The structures of SgGlkA show a dimer-of-dimer assembly. The dimer unit of SgGlkA is shown in 213

Figure 3A. The dimerization interface of SgGlkA is mainly composed of the consensus sequence 1 (residues 214

130–157), α8 helix, and α9-β13 loop. The contact surface area of the SgGlkA dimer in the structure of 215

SgGlkA-glucose is 1943 Å2, which is equivalent to 14.0% of the total surface. The dimerization interface is 216

stabilized by 14 hydrogen-bonds and a hydrophobic contact. When the dimer structures of SgGlkA are 217

compared with those of other ROK family proteins (39, 46, 53), the dimerization mode of each protein is 218

relatively conserved, although the residues in the dimerization interface are not conserved. This might be 219

because the majority of the proton donors or accepters of the intermolecular hydrogen-bonds that are 220

stabilizing the dimerization interface are main chain imide nitrogen or carbonyl oxygen atoms, especially in 221

the consensus sequence 1, and the side chain atoms are not really involved in the intermolecular 222

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hydrogen-bonds. 223

The SgGlkA dimers form a homotetramer using the large α+β domain. In the structure of 224

SgGlkA-glucose, the contact surface area of the SgGlkA tetramerization interface is 571 Å2, which is 225

equivalent to 4.1% of the total surface, and two protomers are stabilized by 10 hydrogen-bonds and 2 226

salt-bridges. Although the dimerization modes of ROK family proteins whose structures have been determined 227

are conserved, these homologous proteins form diverse multimerization forms (Fig. 3DE). In the structure of 228

SgGlkA-glucose, one dimer contacts the other dimer, which rotates counterclockwise 70o around the 229

dimerization axis by head-to-head orientation (Fig. 3B). On the other hand, the transcriptional regulator of the 230

ROK family, Mlc from E. coli, also forms a homotetramer but one dimer comes into contact with the other 231

dimer, which rotates clockwise 45o around the dimerization axis (39). In addition, the N-acetylmannosamine 232

kinase domain of UDP-GlcNac 1-epimerase/ManNAc 6-kinase from humans, which also belongs to the ROK 233

family, forms a homohexamer (53). The homotetrameric structures found in the SgGlkA structures are 234

characteristic of glucokinase from Streptomyces. 235

236

Structure of the Zn-binding motif and glucose-binding mechanism 237

The ROK family proteins possess the highly conserved Zn-binding motif (CXCGXXGCXE) in their 238

consensus sequence 2. Previous structural studies of ROK family proteins have shown that the conserved 239

Zn-binding motif binds a zinc ion (39, 46, 53). The ICP-AES analysis showed that SgGlkA also bound one 240

zinc ion per one SgGlkA molecule (data not shown). Similar to the other ROK family protein structures, three 241

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cysteine residues in the Zn-binding motif (Cys167, Cys169, and Cys174) and His157 in the ExGH motif of 242

SgGlkA form an HC3-type zinc-finger to bind a zinc ion (Fig. 4A) (46, 53). In this study, the structures of 243

SgGlkA-glucose and SgGlkA-glucose-AMPPNP clearly show that H157 in the zinc-finger motif is involved 244

in the glucose-binding mechanism of SgGlkA. 245

The structures of SgGlkA-glucose and SgGlkA-glucose-AMPPNP are the first structures of ROK 246

family proteins in complex with a substrate (Figs. 2 and 4). In each complex, the bound glucose is a cyclic 247

form and adopts the β-anomeric configuration. When the structures of SgGlkA-glucose and 248

SgGlkA-glucose-AMPPNP are compared, the glucose-binding mechanism of the two complexes is found to 249

be identical. SgGlkA binds a glucose molecule at the cleft region between the small domain and the large 250

domain, and recognizes glucose by six residues (Gly66, Asn104, Asp105, Glu154, His157, and Glu176) (Fig. 251

4B). In the small domain, Gly66 in the active site loop of the ROK family proteins (Fig. 1) and Asn104 form 252

hydrogen bonds to C3 hydroxyl of glucose. In addition, Asp105, which is the catalytic Asp of ROK family 253

proteins (Fig. 1), forms a bidentate hydrogen-bond with glucose (C4 and C6 hydroxyls). Larion et al. analyzed 254

D-allose kinase from E. coli (AlsK) (28), which belongs to the ROK family sugar kinases, and found that the 255

Ala73Gly mutant of AlsK exhibited glucokinase activity. The Ala73 of AlsK corresponds to Gly66 of SgGlkA. 256

In the structure of SgGlkA-glucose, Gly66 possesses Gly-specific ψ and φ angles in the Ramachandran plot 257

(118.4o and –172.3o, respectively), and the main chain imide group forms a hydrogen-bond with the C3 258

hydroxyl of glucose. These observations would suggest that the structure of Gly66 is important for the binding 259

of glucose at the active site. 260

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Three of the residues in the large domain, Glu154 and His157 in the ExGH motif, and Glu176 in the 261

Zn-binding motif (Fig. 1), are used to recognize glucose by 5 hydrogen bonds. Glu154 forms a bidentate 262

hydrogen bond with glucose (C2 and C3 hydroxyls). Glu176 forms a hydrogen-bond with C1 hydroxyl. 263

His157, which is involved in the HC3-type zinc-finger, also forms hydrogen-bonds with C1 and C2 hydroxyls 264

of glucose. The orientation of the imidazole ring of His157 is defined by the interaction between the Nδ1 265

nitrogen of His157 and the bound zinc ion. In a study on Bacillus subtilis glucokinase, which belongs to the 266

ROK family, mutations of the cysteine residues in the Zn-binding motif to alanine resulted in a loss of 267

enzymatic activity (35). This would have been because the orientation of His157 was modified so as not to 268

form hydrogen bonds to glucose due to the disruption of the zinc-finger motif. 269

The residues involved in the glucose-binding mechanism are conserved not only in the other ROK 270

family glucokinases but also in the other glucokinase families (Fig. 4B). In EcGlk, which belongs to the group 271

II bacterial glucokinase, Asn99, Asp100, Glu157, Hiss160, and Glu187 are involved in the glucose-binding 272

mechanism by direct hydrogen-bonds (30). When the structures of SgGlkA and EcGlk are compared, the 273

coordinates of the EcGlk residues involved in the glucose recognition are well superposed with those of 274

Asn104, Asp105, Glu154, His157, and Glu176 of SgGlkA, respectively. In addition, the glucose-recognition 275

mechanism is also conserved in a human brain hexokinase I (1). In the human brain hexokinase I, the 276

orientations of glucose-recognizing residues are also conserved, although a Gln residue is substituted for the 277

His157 of SgGlkA that recognizes the C1 and C2 hydroxyls of glucose. Also in the human brain hexokinase I, 278

point-mutations of Asp657, Glu708, and Glu742 lead to the loss of enzymatic activity (5). These residues 279

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correspond to Asp105, Glu154, and Glu176 of SgGlkA. This indicates the importance of these residues for the 280

glucokinase activity. The structural similarity of the glucose-binding site would indicate that the 281

glucose-binding mechanism of glucokinase is evolutionally conserved from bacteria to humans. Meanwhile, 282

Mlc from E. coli, a member of the ROK family, does not have the ability to bind glucose (46). When the structures 283

of SgGlkA and Mlc were compared, Asn104 of SgGlkA, which is involved in the glucose recognition, was replaced 284

by His194 in Mlc. Nε2 and Cε1 atoms of the imidazole ring of His194 may prevent the binding of glucose to Mlc 285

by the conflict with the C3 hydroxyl of glucose. However, a mutation of His194 of Mlc to Asn did not result in 286

glucose binding or glucokinase activity of Mlc (41). The exact reason why Mlc does not bind glucose is still 287

unclear. 288

289

Anomer specificity of SgGlkA 290

The structures of SgGlkA-glucose and SgGlkA-glucose-AMPPNP show that SgGlkA binds the 291

β−anomeric form of glucose, although there is enough space to bind an α-anomer of glucose at the 292

glucose-binding site. Because glucose in solution is a mixture of α- and β-anomers, these observations 293

indicate that SgGlkA prefers to bind the β-anomer of glucose at the active site region. In the structures of 294

SgGlkA-glucose, the C1 hydroxyl group of glucose was recognized by the two hydrogen-bonds with His157 295

and Glu176. Among them, His157 is not conserved in the human brain hexokinase, which prefers to bind the 296

α-anomer of glucose (1). As described above, the orientation of the imidazole ring of His157 is defined by the 297

interaction with the zinc ion bound to the zinc-finger motif. When the coordinates of the hydrogen atom 298

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bound to Nε2 nitrogen are predicted, the distance between the C1 hydroxyl of β-glucose and the Nε2 299

hydrogen of SgGlkA is 2.29 Å. On the other hand, when the putative C1 hydroxyl position of α-glucose is 300

predicted, the C1 hydroxyl of α-glucose exists relatively far from the Nε2 hydrogen of SgGlkA (2.75 Å) (Fig. 301

4C). This difference would relate to the β-anomer selectivity of SgGlkA. 302

303

Nucleotide binding in the active site 304

SgGlkA possesses a DxGxT-type ATP-binding motif in its N-terminal region (Fig. 1) (18, 28). 305

During the structure determination of SgGlkA-glucose-AMPPNP, the Fo-Fc difference map in the active site 306

cleft region shows the presence of AMPPNP around the glucose-binding site (Fig. 2B). Unexpectedly, the 307

electron density of the γ-phosphate group of AMPPNP was not observed. In some cases, it is known that 308

AMPPNP is hydrolyzed into a distinct product similar to ADP (ADPβN) (32, 49). The γ-phosphate of 309

AMPPNP in the SgGlkA-glucose-AMPPNP complex might also be dephosphorylated during the 310

crystallization processes. Although the crystallization condition of SgGlkA-glucose-AMPPNP contained 5 311

mM MgCl2, we could not observe any electron density of Mg2+ ions around the AMPPNP. The AMPPNP 312

molecule is located between the large domain and the small domain, and is recognized by eight 313

hydrogen-bonds (Fig. 4D). The adenine moiety of AMPPNP is recognized by two hydrogen-bonds with two 314

residues of the large domain, Ser219 and Glu265. The α-phosphate group of AMPPNP forms two 315

hydrogen-bonds with Lys13 and Gly261. The β-phosphate group of AMPPNP is pinched by two loops (β1-β2 316

and β9-β10) and recognized by four hydrogen-bonds. Around the ATP-binding motif, two main chain imides 317

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of Thr12 and Lys13 form two hydrogen-bonds with the β-phosphate group. In addition, a main chain imide 318

and a side chain hydroxyl of Thr132 also form two hydrogen-bonds with the β-phosphate group. 319

In the structure of SgGlkA-glucose-AMPPNP, the distance between the β-phosphorus and C6 320

hydroxyl of glucose is 5.97 Å (Fig. 4E). This is close enough to transfer the γ-phosphate group of ATP to the 321

C6 hydroxyl. In the analysis of human brain hexokinase I, mutations of Asp532 in its ATP-binding motif, 322

which structurally corresponds to Asp8 of SgGlkA, to Lys or Glu results in a significant loss of the enzymatic 323

activity (58). In the structure of SgGlkA-glucose-AMPPNP, Asp8 locates between the AMPPNP and Asn105, 324

and seems to recognize the γ-phosphate group of ATP. Asp8 of SgGlkA would also be important for the 325

kinase activity. 326

327

Structural modification depending on the substrate binding 328

Glucokinase may be a key player in CCR in Streptomyces (3, 4, 27, 31). In this structural study, we 329

have determined three structures, apo-SgGlkA, SgGlkA-glucose, and SgGlkA-glucose-AMPPNP. The 330

structures of apo-SgGlkA and SgGlkA-glucose-AMPPNP may represent the structures of SgGlkA in a cell 331

lacking and rich in glucose and ATP, respectively. Like the structures of the other ROK family proteins and 332

other glucokinases (30, 39), the structure of SgGlkA is influenced by the binding of its substrates. 333

In the crystal of apo-SgGlkA, eight molecules of SgGlkA were contained in its asymmetric unit and 334

formed two SgGlkA tetramers. When the chain A structure of apo-SgGlkA is compared with those of chains 335

B-H, the root-mean-square-deviation (r.m.s.d.) values of the superposed Cα atoms are 0.4 – 0.6 Å. This result 336

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indicated that the structures of the apo-SgGlkA protomers are approximately the same in the asymmetric unit. 337

On the other hand, when the structures of apo-SgGlkA and SgGlkA-glucose are compared, the structures are 338

not identical. By the analysis using the program DynDom (15), the structure of the small domain rotates 6.2o 339

toward the large domain by the binding of glucose (Fig. 5A). The r.m.s.d value between the chain A structure 340

of apo-SgGlkA and SgGlkA-glucose for the superposed 311 Cα atoms is 1.1 Å. During the conformational 341

change, the structure of the β4-β5 loop (active site loop in Fig. 1), which includes the glucose recognizing 342

Gly66, approaches that of the bound glucose. By this structural change, the Cα position of Gly66 moves 2.08 343

Å toward the large domain (Fig. 5B). Because the coordinates of the residues involved in the glucose-binding 344

mechanism are not largely modified, except in the case of Gly66, the intermolecular interaction between the 345

bound glucose and Gly66 would be the key factor for the glucose-dependent conformational change of 346

SgGlkA. Meanwhile, when the structures of SgGlkA-glucose and SgGlkA-glucose-AMPPNP are compared, 347

the structure of the small domain rotates 12.3o toward the large domain. The r.m.s.d. value between the 348

structures of SgGlkA-glucose and SgGlkA-glucose-AMPPNP for the superposed 312 Cα atoms is 1.7 Å. This 349

conformational change is induced by the binding of AMPPNP. As described above, the β-phosphate group of 350

the AMPPNP is recognized by the two loops, β1-β2 and β9-β10. By the binding of AMPPNP, the β1-β2 loop 351

approaches the β9-β10 loop to pinch the β-phosphate group (Fig. 5C). By this structural change, the main 352

chain imide of Thr12, which forms the hydrogen-bond with AMPPNP, moves 5.23 Å toward the large 353

domain. 354

In the analysis of mammalian glucokinases, the activities of the enzymes exhibit a sigmoidal glucose 355

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dependency instead of the Michaelis-Menten hyperbolic dependency, although they are monomeric enzymes 356

(25, 48). The “allosteric” properties, which enable mammalian glucokinases to play the role of a glucose 357

sensor in the pancreas and liver, result from the conformational change of the enzyme by the binding of 358

glucose (25). At low glucose concentrations, human glucokinase adopts a low-affinity structure (super-open 359

form) demonstrating a “slow” catalytic cycle. When glucose concentrations are sufficiently high, it adopts a 360

high-affinity structure (open form) showing a “fast” catalytic cycle. This shift of the major catalytic cycle 361

explains the sigmoidal saturation curve for glucose. To examine whether SgGlkA is also allosterically 362

activated by glucose, SgGlkA activity (the SgGlkA-catalyzed rate of glucose phosphorylation, v (mM min-1)) 363

was measured at various glucose concentrations, S (0.125-5 mM) and two fixed ATP concentrations (4.3 and 364

0.86 mM). In the Lineweaver-Burk plot (1/v vs. 1/[S]), two linear lines with a similar x-intercept were 365

obtained (Fig. 6A), indicating that the SgGlkA activity exhibited the Michaelis-Menten hyperbolic 366

dependency with a Michaelis constant (Km) for glucose, independently of the ATP concentrations. This result 367

was consistent with the kinetic studies of glucokinases from Streptomyces coelicolor A3(2) and Streptomyces 368

peucetius var. caesius (20), which indicated that their kinetic mechanism followed a rapid equilibrium-ordered 369

Bi-Bi sequential mechanism (they first bind glucose and then the MgATP2+ complex). We determined the 370

kinetic parameters from the SgGlkA activity in the presence of an excess amount of ATP (4.3 mM) as shown 371

in Fig. 6B. It should be noted that the Vmax of SgGlkA (0.935 ± 0.049 μmol min-1 mg-1) is broadly comparable 372

to the Vmax values of S. coelicolor glucokinase (1.6667 ± 0.170 μmol min-1 mg-1) and S. peucetius glucokinase 373

(1.0867 ± 0.0286 μmol min-1 mg-1) (20). From these results, we concluded that SgGlkA does not exhibit a 374

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sigmoidal glucose dependency, in spite of the structural similarity between the human glucokinase (PDB code: 375

1V4S) and SgGlkA-glucose (r.m.s.d. = 3.1 Å, number of Cα−atoms superposed = 282). This result indicates 376

that the conformational change observed in the structures of SgGlkA does not relate to the activity regulation 377

of this enzyme. Because SgGlkA does not have a DNA-binding motif, unlike ROK family transcriptional 378

regulators such as Mlc, SgGlkA would not be able to directly regulate the transcriptions of CCR-related genes 379

(39, 46). Nonetheless, the conformational change in the presence of substrates could be a mechanism by 380

which SgGlkA transmits the existence of glucose to other proteins that regulate the CCR of Streptomyces. 381

Although the SgGlkA-dependent catabolite repression mechanism in Streptomyces is still unclear, the 382

substrate-dependent conformational changes of SgGlkA observed in this study may play a role as a glucose 383

sensor in this organism. 384

385

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Acknowledgments 386

387

The synchrotron-radiation experiments were performed at beamline BL-17A of the Photon Factory 388

(Proposal No. 2008S2-001). This work was supported by the Targeted Proteins Research Program (TPRP) of 389

the Ministry of Education, Culture, Sports, Science, and Technology, Japan. 390

391

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525

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Figure legends 526

527

Figure 1. Amino acid sequence alignment of SgGlkA with its homologous ROK family proteins. Secondary 528

structure assignments of SgGlkA are indicated by helices (α- and 310 (η)- helices), arrows (β-strands), and TT 529

(β-turns). Strictly conserved residues among the proteins are shown with a red background. The protein names 530

are indicated by the PDB accession numbers as follows: 2AA4, putative N-acetylmannnosamine kinase from 531

E. coli; 2QM1, glucokinase from Enterococcus faecalis; 1Z6R, Mlc from E. coli. Important regions of the 532

ROK family proteins (ATP-binding motif, active site loop, catalytic asp, ExGH motif, and Zn-binding motif) 533

and consensus sequences 1 & 2 (CS1 and CS1) are annotated and delineated by a line. Residues involved in 534

the glucose-binding mechanism are indicated by asterisks. 535

536

Figure 2. Protomer structure of SgGlkA. (A) Protomer structure of SgGlkA-glucose shown with a ribbon 537

diagram. Color-coding runs from blue in the N-terminus to red in the C-terminus. Secondary structure 538

assignments are labeled on the ribbon diagram. The glucose molecule is shown by a stick model. The zinc ion 539

is shown by a gray sphere model. (B) Protomer structure of SgGlkA-glucose-AMPPNP. An Fo-Fc omit map of 540

the glucose and AMPPNP contoured at 2.0 σ is shown as a blue mesh. The ATP-binding motif, active site loop, 541

catalytic Asp, consensus sequence 1, and consensus sequence 2 are colored red, orange, blue, green, and cyan, 542

respectively. 543

544

Figure 3. Multimerization of SgGlkA. (A) Dimer structure of SgGlkA-glucose. One protomer is shown by a 545

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rainbow-colored ribbon and by a cyan surface, and the other is shown by a gray ribbon and surface. (B) 546

Tetramer structure of SgGlkA-glucose. Protomers are shown by cyan, gray, orange, and light blue surfaces. 547

(C) Oligomeric state analyses of apo-SgGlkA and SgGlkA-glucose by gel filtration chromatography. The 548

results for the standard proteins aldolase (Mr 158,000), canalbumin (Mr 75,000), ovalbumin (Mr 43,000), and 549

carbonic anhydrase (Mr 29,000) are indicated by triangles. (D) Tetrameric structure of E. coli Mlc (PDB ID: 550

3BP8). The respective protomers are colored cyan, white, orange, and purple. (E) Hexameric structure of the 551

N-acetylmannosamine kinase domain of the UDP-GlcNac 1-epimerase/ManNAc 6-kinase (GNE) from a 552

human (PDB ID: 3EO3). The respective protomers are colored cyan, white, orange, purple, green, and 553

magenta. 554

555

Figure 4. Substrate-binding mechanisms of SgGlkA. (A) HC3-type zinc-finger motif formed by the residue in 556

the ExGH motif (His157) and Zn-binding motif (Cys167, Cys169, and Cys174). (B) The superposition of the 557

glucose-binding sites. The superposition was performed using the coordinates of the glucose molecules except 558

for the C1 hydroxyls. Green, magenta, and cyan stick models show the structures of SgGlkA-glucose, EcGlk 559

(PDB ID: 1SZ2), and human brain hexokinase I (PDB ID: 1DGK), respectively. Residue numbers of SgGlkA 560

and the distances of hydrogen-bonds are indicated in black letters. The hydrogen-bonds in the structure of 561

SgGlkA-glucose are shown by blue dotted lines. (C) Putative hydrogen-bond between C1 hydroxyl of 562

α-glucose (gray) and Nε2 hydrogen of His157. A green stick model shows the structure of SgGlkA-glucose. 563

The distances between the Nε2 hydrogen of His157 and C1 hydroxyls of glucose are indicated in black letters. 564

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(D) AMPPNP-binding mechanism observed in the structure of SgGlkA-glucose-AMPPNP. The bound 565

AMPPNP and the residues involved in the ATP-binding mechanism are shown by stick models. (E) Structure 566

around the AMPPNP and glucose. The distance between the β-phosphorus atom of AMPPNP and the C6 567

hydroxyl of glucose is 5.97 Å. 568

569

Figure 5. Conformational changes of SgGlkA by the binding of substrates. (A) Conformational change of the 570

SgGlkA protomer. The structures of apo-SgGlkA, SgGlkA-glucose, and SgGlkA-glucose-AMPPNP are 571

colored green, white, and magenta, respectively. Three structures are superposed using the coordinates of Cα 572

atoms of the large domain. (B) Conformational change by the binding of glucose. The distance between the 573

Cα atoms of Gly66 in the structures of apo-SgGlkA and SgGlkA-glucose is 2.08 Å. (C) Conformational 574

change by the binding of AMPPNP. Thr12 moves 5.23 Å toward the large domain. 575

576

Figure 6. Kinetic analysis of SgGlkA 577

(A) Lineweaver-Burk plot of the SgGlkA activity (the SgGlkA-catalyzed rate of glucose phosphorylation, v 578

(nmol sec-1)) at various glucose concentrations, S (mM), and two fixed ATP concentrations (4.3 and 0.86 mM). 579

The data obeyed the Michaelis-Menten equation. (B) Kinetic parameters of SgGlkA. The values are shown as 580

the average values from three independent experiments with standard deviations. 581

582

583

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TABLE 1.

Summary of data collection and refinement statistics for SgGlkA crystals

SgGlkA SgGlkA-glucose SgGlkA-glucose-AMPPNP

Data collection Beam line PF BL-17A PF BL-17A PF BL-17A

Space group P21 P6422 I222 Cell dimensions a, b, c (Å) 98.69, 173.70, 124.03 108.19, 108.19, 141.18 69.58, 87.73, 125.39

β (ο) 106.69

Wavelength (Å) 1.00000 1.00000 1.00000 Resolution (Å) 20.0-3.25 (3.33-3.25)* 20.0-1.84 (1.89-1.84) 20.0-1.55 (1.59-1.55) Rmerge (%) 11.3 (82.9) 4.1 (33.0) 5.1 (34.6)

I/σI 10.15 (2.14) 32.89 (4.31) 20.14 (2.68)

Completeness (%) 96.3 (97.6) 99.8 (98.9) 96.0 (70.9) Redundancy 4.1 (4.0) 9.7 (5.3) 6.0 (2.3)

Refinement

R/Rfree (%) 21.2/26.5 16.8/18.5 17.9/20.2 Number of atoms Protein 18132 2271 2271 Ligand 0 12 39

Ions 18 2 2 Water 0 253 278 R.M.S deviations Bond lengths (Å) 0.014 0.014 0.011

Bond angles (ο) 1.448 1.286 1.308 Ramachandran plot Favored region (%) 94.2 99.0 99.4 Allowed region (%) 5.8 1.0 0.6

Outer region (%) 0.0 0.0 0.0

*Values in parenthesis are for the highest resolution shell.

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