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J. Biol. Chem., Vol. 282, Issue 13, 9923-9931, March 30, 2007

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Crystal Structures of an ATP-dependent Hexokinase with Broad Substrate Specificity from the Hyperthermophilic Archaeon Sulfolobus tokodaii^*

From the Department of Biotechnology, the University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received for publication, November 17, 2006 , and in revised form, December 27, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Hexokinase catalyzes the phosphorylation of glucose to glucose 6-phosphate by using ATP as a phosphoryl donor. Recently, we identified and characterized an ATP-dependent hexokinase (StHK) from the hyperthermophilic archaeon Sulfolobus tokodaii, which can phosphorylate a broad range of sugar substrates, including glucose, mannose, glucosamine, and N-acetylglucosamine. Here we present the crystal structures of StHK in four different forms: (i) apo-form, (ii) binary complex with glucose, (iii) binary complex with ADP, and (iv) quaternary complex with xylose, Mg²⁺, and ADP. Forms i and iii are in the open state, and forms ii and iv are in the closed state, indicating that sugar binding induces a large conformational change, whereas ADP binding does not. The four different crystal structures of the same enzyme provide "snapshots" of the conformational changes during the catalytic cycle. StHK exhibits a core fold characteristic of the hexokinase family, but the structures of several loop regions responsible for substrate binding are significantly different from those of other known hexokinase family members. Structural comparison of StHK with human N-acetylglucosamine kinase and other hexokinases provides an explanation for the ability of StHK to phosphorylate both glucose and N-acetylglucosamine. A Mg²⁺ ion and coordinating water molecules are well defined in the electron density of the quaternary complex structure. This structure represents the first direct visualization of the binding mode for magnesium to hexokinase and thus allows for a better understanding of the catalytic mechanism proposed for the entire hexokinase family.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Phosphorylation of glucose to glucose 6-phosphate is important for both energy metabolism and biosynthesis in the cell. In eukaryotes, the reaction is catalyzed by hexokinases (EC 2.7.1.1 [EC] ), which can phosphorylate several hexoses, including mannose and fructose, in addition to glucose (1). In contrast, bacteria possess glucokinases (EC 2.7.1.2 [EC] ) that are specific for glucose. Bacterial glucokinases can be classified into two groups as follows: (i) glucokinases belonging to the repressors/open reading frames of unknown function/sugar kinases (ROK)² family, which is characterized by two signature motifs (2), and (ii) glucokinases without the ROK motifs (3). On the other hand, most Archaea use two types of glucokinase as follows: (i) ADP-dependent glucokinases (4–9), or (ii) ATP-dependent glucokinases belonging to the ROK family (10, 11).

On the basis of amino acid sequence similarity, sugar kinases can be divided into three families as follows: (i) hexokinase family, (ii) galactokinase family, and (iii) ribokinase family (12). Hexokinases and glucokinases belong to the hexokinase family, with the exception of the archaeal ADP-dependent glucokinases (13–15), which are members of the ribokinase family. The crystal structures of several members of the hexokinase family have been reported, including human hexokinase (16–20), rat and Schistosoma mansoni hexokinase (21), yeast hexokinase (22–25), Escherichia coli glucokinase (26), Arthrobacter sp. strain KM glucomannokinase (27), and E. coli rhamnulose kinase (28). These molecules possess an identical core structure consisting of two domains with the fold and the active site located between the two domains. Two -turns formed by the conserved DXGGT and GTG motifs are involved in ATP binding. The hexokinase family is included in the ASKHA (acetate and sugar kinases/Hsp70/actin) superfamily (29, 30), the members of which have a common core fold and catalyze phosphoryl transfer or hydrolysis of ATP.

In the past, ATP-dependent glucose phosphorylating activity had been detected in cell extracts of the hyperthermophilic archaeon Sulfolobus solfataricus (31), but no homologs of known hexokinases and glucokinases have been found in the genomes of the Sulfolobus species (32–34). Recently, we purified the ATP-dependent glucose phosphorylating activity from cell extracts of Sulfolobus tokodaii and identified the gene responsible for the activity (35). Our kinetic analyses indicated that S. tokodaii hexokinase (StHK) is a novel hexokinase that can phosphorylate not only glucose but also GlcNAc, glucosamine, and mannose. The enzyme differs from other known hexokinases and glucokinases in that its activity is strongly inhibited by ADP. StHK shows 25% amino acid sequence identity with mammalian GlcNAc kinases (supplemental Fig. S1). However, StHK is distinct in its broad substrate specificity from the GlcNAc kinases (36, 37), which are specific for GlcNAc. Although mammalian GlcNAc kinases show no significant sequence similarity with hexokinases, they possess the two ATP-binding motifs (EGGGT and GTG) characteristic of the hexokinase family and are therefore classified into this family (38). Very recently, the crystal structures of human GlcNAc kinase in complex with GlcNAc and with glucose and ADP have been reported (39). The structures reveal that human GlcNAc kinase has a similar core fold to the hexokinase family. StHK also contains the DAGGT and GTG motifs at the amino-terminal and middle regions of the sequence, respectively (supplemental Fig. S1), indicating that StHK is a new member of the hexokinase family with unique substrate specificity.

Here we report the crystal structures of StHK in four different forms as follows: (i) the apo-form; (ii) complex with glucose; (iii) complex with ADP; and (iv) complex with xylose, Mg²⁺, and ADP. These structures provide a molecular basis for the substrate specificity, conformational changes upon substrate binding, and catalytic mechanism of this enzyme.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Sample Preparation and Crystallization—StHK was expressed in E. coli and purified to homogeneity as described (35). The selenomethionine-substituted protein was expressed in E. coli B834 (DE3). The cells were grown at 37 °C for 20 h in 1 liter of SeMet core medium (Wako) supplemented with 10 g of glucose, 0.25 g of MgSO₄·7H₂O, 4 mg of FeSO₄·7H₂O, 10 ml of vitamin growth supplement (Sigma), 0.1 g of ampicillin, and 25 mg of seleno-L-methionine (Wako). The protein was purified using the same protocol as for the native protein. The native and selenomethionine-substituted proteins were concentrated to 15 mg/ml in 10 mM Tris-HCl (pH 8.0) and used for crystallization.

Crystallization was performed at 25 °C using the hanging drop vapor diffusion method. Crystals of the apo-form were grown by mixing 2 µl of the protein solution and 1 µl of the reservoir solution consisting of 0.1 M HEPES-NaOH (pH 7.5) and 1.3 M Li₂SO₄. Crystals of the ADP complex were grown by mixing 2 µl of the protein solution containing 5 mM ADP and 10 mM MgCl₂, and 1 µl of the reservoir solution consisting of 0.1 M HEPES-NaOH (pH 7.7) and 1.3 M Li₂SO₄. Crystals of the glucose complex were grown by mixing 2 µl of the protein solution containing 10 mM glucose and 0.2 M MgCl₂, and 1 µl of the reservoir solution consisting of 0.1 M Tris-HCl (pH 8.6) and 34% PEG3350. Crystals of the xylose·Mg²⁺·ADP complex were grown by mixing 2 µl of the protein solution containing 10 mM xylose, 20 mM MgCl₂, and 10 mM ATP, and 1 µl of the reservoir solution consisting of 0.1 M Tris-HCl (pH 9.0) and 18% PEG3350. The selenomethionine-substituted protein was crystallized under conditions similar to those used for the native crystals of the glucose complex. All crystals grew to full size within 1 week.

Data Collection—X-ray diffraction data were collected using a charged-coupled device (CCD) camera at the BL-5 and BL-6A station at the Photon Factory, and the NW12 station at the Photon Factory AR, High Energy Accelerator Research Organization (KEK), Tsukuba, Japan. Crystals of the apo-form were cryoprotected in the reservoir solution supplemented with 20% trehalose. Crystals of the ADP complex were cryoprotected in the reservoir solution supplemented with 5 mM ADP, 10 mM MgCl₂, and 20% trehalose. Crystals of the xylose·Mg²⁺·ADP complex were cryoprotected in the reservoir solution supplemented with 10 mM xylose, 20 mM MgCl₂, 10 mM ATP, and 15% PEG400. All crystals were flash-frozen at 100 K in a stream of liquid nitrogen. Data were processed using the HKL2000 (40).

Structure Determination and Refinement—Initially, the structure of the glucose complex was determined by multiwavelength anomalous diffraction using a selenomethionine-substituted crystal. All eight selenium atoms were located, and initial phases were calculated using SOLVE/RESOLVE (41), followed by automated model building using ARP/wARP (42). The resultant model was refined against the native data set of the glucose complex. Manual model building and refinement were performed using XtalView (43) and CNS (44), respectively. The structure of the glucose complex was refined at 1.65 Åtoan R factor = 18.8 (R_free = 21.8). The crystal belongs to space group C222₁ with one molecule in the asymmetric unit. The final model contains residues 1–299, 1 glucose molecule, and 375 water molecules.

The structure of the apo-form was determined at 1.9 Å resolution by molecular replacement using MOLREP (45) in CCP4 (46) with the small and large domains of the glucose complex structure as search models, and refined to an R factor = 17.5 (R_free = 21.0). The crystal belongs to space group P2₁ with two molecules in the asymmetric unit. The final model contains molecules A (residues 1–298) and B (residues 1–297) as well as three sulfate ions, one HEPES molecule, and 573 water molecules. The two sulfate ions are bound at the dimer interface, whereas the other sulfate ion and the HEPES molecule are located far from the active site allowing them to participate in crystal packing interactions. As the two molecules are essentially identical (r.m.s.d. for C- atoms = 0.44 Å), molecule A is described as the apo structure.

The structure of the ADP complex was determined at 2.0 Å resolution by molecular replacement and refined to an R factor = 18.3 (R_free = 22.6). The crystal belongs to space group P2₁ with two molecules in the asymmetric unit. The final model contains molecule A (residues 1–298) and B (residues 1–297) as well as two ADP molecules, three sulfate ions, one HEPES molecule, and 482 water molecules. As the two molecules are essentially identical (r.m.s.d. for C- atoms = 0.38 Å), molecule A is described as the ADP complex structure.

The structure of the xylose·Mg²⁺·ADP complex was determined at 2.0 Å resolution by molecular replacement and refined to an R factor = 21.3 (R_free = 25.5). The crystal belongs to space group C2 with two molecules in the asymmetric unit. The final model contains molecule A (residues 1–297) and B (residues 1–296) in addition to one xylose molecule, one Mg²⁺ ion, two ADP molecules, and 261 water molecules. Two ADP molecules are included in the model, because no electron density was observed for the -phosphate of ATP, probably because of hydrolysis of ATP during crystallization, although the protein was crystallized in the presence of ATP. Molecule A adopts a closed conformation with bound xylose, Mg²⁺, and ADP, whereas molecule B adopts an open conformation with bound ADP and is essentially identical to the ADP complex structure (r.m.s.d. for C- atoms = 0.66 Å). Molecule A is described as the xylose·Mg²⁺·ADP complex structure.

Data collection and refinement statistics are shown in Table 1. Figures were prepared using PyMol (47).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Apo-form—The structure of StHK (299 amino acid residues) in the apo-form was determined at 1.9 Å resolution. The structure consists of two domains, each of which has the fold characteristic of the ASKHA superfamily (Fig. 1A). The small domain (residues 1–107 and 274–299) is composed of a five-stranded mixed -sheet (3, 2, 1, 4, and 5), with strand 2 antiparallel to the rest, flanked on one side by a pair of -helices (2 and 3) and on the other by helix 11. The large domain (residues 108–273) is composed of a five-stranded mixed -sheet (8, 7, 6, 9, and 10), with strand 7 anti-parallel to the rest, six -helices (5 to 10), and a 3₁₀-helix. The -sheet is flanked on one side by helix 4 and on the other side by a pair of -helices (9 and 10). The two domains are connected by helix 4. An intramolecular disulfide bond is formed by the conserved cysteine residues Cys-21 and Cys-291.

A structural similarity search using Dali (48) indicated that StHK shows relatively high degrees of similarity to the hexokinase family members, including E. coli glucokinase (26) (Protein Data Bank code 1Q18 [PDB] ; Z score = 15.9, and root mean square deviation (r.m.s.d.) for 237 C- atoms = 3.6 Å), Arthrobacter sp. strain KM glucomannokinase (27) (Protein Data Bank code 1WOQ [PDB] ; Z score = 15.0, and r.m.s.d. for 205 C- atoms = 3.2 Å), the carboxyl-terminal catalytic domain (residues 525–917) of human hexokinase I (18) (Protein Data Bank code 1QHA [PDB] ; Z score = 15.5, and r.m.s.d. for 241 C- atoms = 3.4 Å), and yeast hexokinase PII (23) (Protein Data Bank code 2YHX [PDB] ; Z score = 13.3, and r.m.s.d. for 228 C- atoms = 3.7 Å). StHK also shows modest structural similarity with other members of the ASKHA superfamily, such as E. coli glycerol kinase (49) (Protein Data Bank code 1GLC; Z score = 13.6, and r.m.s.d. for 235 C- atoms = 5.0 Å). The closest structural relative is the recently solved human GlcNAc kinase complexed with GlcNAc (39) (Protein Data Bank code 2CH5; Z score = 28.1, and r.m.s.d. for 290 C- atoms = 3.1 Å). The secondary structures are conserved within the two enzymes except that human GlcNAc kinase has an additional -strand at the carboxyl terminus to form a six-stranded mixed -sheet.

View larger version (62K): [in this window] [in a new window]   FIGURE 1. Overall structures of StHK. A, stereo view of the monomer structure in the apo-form. The small and large domains are colored cyan and pink, respectively. The 1-2 and 6-7 turns are colored dark blue. The disulfide bond between Cys-21 and Cys-291 is shown in stick representation. B, stereo view of the dimer structure in complex with glucose. The bound glucose is shown in stick representation with carbon atoms in yellow. The region involved in dimer formation is colored green. The adjacent subunit is colored gray.

Glucose Complex—StHK was crystallized in the presence of glucose, and the glucose complex structure was determined at 1.65 Å resolution. Clear electron density for glucose is observed at the pocket formed by four loops, the 3-1 and 4-3 loops of the small domain and the 5-4 and 8-5 loops of the large domain (Fig. 2A). As observed in E. coli glucokinase (26), glucose is bound in the -anomeric configuration. The hydroxyl groups of glucose form direct hydrogen bonds with Gly-69, Asp-71, His-94, Asp-95, Gly-135, and Asp-140 and water-mediated hydrogen bonds with Gly-11, Asn-35, and Gly-117. In addition, the 2-hydroxyl group is hydrogen-bonded with Tyr-189 of the adjacent subunit. The extensive hydrogen bonding interactions between the enzyme and glucose can explain the high affinity of the enzyme for glucose (K_m = 0.050 mM) (35). These residues involved in glucose binding are completely conserved in the homologs from Sulfolobus species (supplemental Fig. S1).

ADP Complex—The enzyme was crystallized in the presence of Mg²⁺ and ADP, and the structure was determined at 2.0 Å resolution. As no electron density was observed for a Mg²⁺ ion in this form, we call it the "ADP complex" structure. Clear electron density for ADP is observed in the cleft of the large domain formed by helices 5, 8, and 3₁₀ and a turn between 6 and 7 (Fig. 2B). The ribose moiety adopts the C2'-endo pucker, and the adenine ring is in the anti-conformation. The adenine ring is sandwiched by the side chains of Lys-203 and Arg-251, with the N-6 atom interacting with the carboxyl group of Asp-206 though a hydrogen bond. The 2'-hydroxyl group of the ribose forms a hydrogen bond with the backbone carbonyl of Ala-199. The 3'-hydroxyl group of the ribose forms a water-mediated hydrogen bond with the backbone carbonyl of Asp-196. In addition, the ribose ring makes van der Waals interactions with the side chains of Ala-144, Ala-202, and Met-249. On the other hand, the - and -phosphoryl groups are recognized by the 3₁₀-helix and the turn between 6 and 7. The -phosphoryl group interacts with the backbone amide of Gly-248 and the side chain of Arg-251. The -phosphoryl group forms hydrogen bonds with the backbone amide and the hydroxyl group of Thr-116. A number of interactions between the enzyme and ADP can explain the high affinity of the enzyme for ATP (K_m = 0.12 mM) in addition to the strong competitive inhibition by ADP (K_i = 18 µM) (35).

Xylose·Mg²⁺·ADP Complex—The structure of StHK complexed with xylose, Mg²⁺, and ADP (xylose·Mg²⁺·ADP) was determined at 2.0 Å resolution. All of the ligands, as well as five water molecules coordinating the Mg²⁺ ion, are well defined in the electron density map (Fig. 2C). As with other kinases, Mg²⁺ is required for the enzyme activity of StHK (35). In the complex structure, the Mg²⁺ ion is octahedrally coordinated by a -phosphoryl oxygen of ADP and five water molecules. Asp-8, Lys-15, and Asp-95 form hydrogen bonds with the water molecules coordinating the Mg²⁺ ion. One of the five water molecules coordinating the Mg²⁺ ion does not interact with the protein but forms a hydrogen bond with the -phosphate of ADP.

View larger version (64K): [in this window] [in a new window]   FIGURE 2. Stereo view of the active sites of the glucose complex (A), the ADP complex (B), and the xylose·Mg2+·ADP complex (C). Residues interacting with ligands are shown in stick representation with carbon atoms colored as in Fig. 1. Ligands are shown in stick representation with carbon atoms in yellow. Water molecules are shown as red spheres. Hydrogen bonds are shown as dashed lines. The Fo - Fc omit electron density maps for ligands are shown as cyan meshes and contoured at 6 (A) and 5 (B and C). A, Tyr-189 from the adjacent subunit is shown in gray and indicated by a prime. C, the Mg2+ ion is shown as a green sphere.

In the complex structure, the hydroxyl groups of xylose are recognized by the enzyme in a manner similar to that in the glucose complex, except for the absence of the hydrogen bond between the enzyme and the 6-hydroxyl group of glucose. The binding mode between the enzyme and the AMP portion of ADP is also similar to that in the ADP complex. In contrast, the binding mode between the enzyme and the -phosphate in the xylose·Mg²⁺·ADP complex is remarkably different from that in the ADP complex. The -phosphate interacts with the backbone amides of Gly-11, Thr-12, and Lys-13 in the 1-2 turn in addition to the backbone amide and the hydroxyl group of Thr-116 in the 6-7 turn. The additional interactions result from the closed conformation of the xylose·Mg²⁺·ADP complex (described below). The two -turns involved in ATP binding are a hallmark of the ASKHA superfamily (29, 30).

View larger version (46K): [in this window] [in a new window]   FIGURE 3. Stereo view of the superposition of the apo-form (gray), the glucose complex (green), and the xylose·Mg2+·ADP complex (magenta) based on the large domains. Ligands are shown in stick representation. The Mg2+ ion is shown as a magenta sphere. Water molecules involved in the hydrogen bonding interactions between the small and large domains of the glucose complex are shown as green spheres. The 1-2 turn is indicated by blue arrows. The ADP complex structure is not shown because it is essentially identical to the apo structure.

The xylose·Mg²⁺·ADP complex structure can be superimposed with the glucose complex structure with r.m.s.d. for C- atoms of 0.79 Å (Fig. 3). However, local structural differences are observed between them. In the xylose·Mg²⁺·ADP complex, the 1-2 turn moves slightly away from the 6-7 turn to accommodate the phosphoryl groups of ADP. The C- atom of Thr-12 in the 1-2 turn shifts by 1.7 Å from its position in the glucose complex. In addition, the side chain of Lys-15, which forms a salt bridge with Glu-278 in the glucose complex, is directed toward ADP to interact with the -phosphoryl group. The glucose complex crystals cracked and dissolved when soaked in an ADP-containing solution, supporting the notion that additional conformational changes would occur upon ADP binding from the closed conformation stabilized by glucose binding.

The apo and ADP complex structures are essentially identical (r.m.s.d. for C- atoms = 0.17 Å), indicating that ADP can bind to the apoenzyme without causing conformational changes in the enzyme. This was supported by our observation that the apo crystals were stable when soaked in an ADP-containing solution and its remarkable inhibition by ADP, in marked contrast to other members of the hexokinase family. No crystal structure bound to only ADP has been reported among other members of the hexokinase family, although a number of crystal structures of these enzymes bound only to glucose or to both glucose and ADP have been solved. For example, it has been reported that the co-crystal structure of E. coli glucokinase with ADP could not be obtained in the presence or absence of glucose (26), suggesting that the enzyme in the open conformation may have low affinity for ADP. In the E. coli enzyme, conformational changes from the open to closed state have been suggested to be a prerequisite for ATP binding, and the activity of the enzyme has not been reported to be inhibited by ADP.

Substrate Specificity—In the ASKHA superfamily, substrate specificity of each member is thought to be mediated by diverged insertions into the conserved core fold (30). The crystal structures of StHK represent the first example of a sugar kinase that can phosphorylate both glucose and GlcNAc efficiently, and thus provide new insights into substrate recognition.

Comparison of StHK with human GlcNAc kinase explains why StHK can phosphorylate not only glucose but also GlcNAc. As shown in Fig. 4A, in the structure of human GlcNAc kinase in complex with GlcNAc, the carbonyl oxygen of the N-acetyl moiety of GlcNAc forms a hydrogen bond with N- of Asn-36, and its methyl group is recognized by a hydrophobic pocket formed by Trp-38 and Trp-146, and Leu-201 and Tyr-205 from the adjacent subunit (39). Asn-35, His-37, Arg-134, Val-185, and Tyr-189 of StHK correspond to Asn-36, Trp-38, Trp-146, Leu-201, and Tyr-205 of human GlcNAc kinase, respectively, suggesting that in StHK these residues are involved in binding the N-acetyl moiety of GlcNAc. However, modeling of GlcNAc into the active site of the glucose complex of StHK, simply based on the position of glucose, indicates that there would be steric hindrance between the N-acetyl moiety and the side chains of Asp-71 and Tyr-189. The side chain of Tyr-189 may rotate to form a hydrophobic interaction with the GlcNAc methyl group. Superposition of the glucose complex of StHK and the GlcNAc complex of human GlcNAc kinase, based on the large domains, indicates that StHK adopts a more closed conformation relative to human GlcNAc kinase (Fig. 4B). This suggests that StHK may adopt a more open conformation when it accommodates GlcNAc. StHK was also crystallized in the presence of GlcNAc using a crystallization buffer similar to that used for the glucose complex, but the crystals did not diffract to high resolution. This result supports the assumption that the GlcNAc complex of StHK would adopt a similar but not identical conformation to that of the glucose complex.

View larger version (64K): [in this window] [in a new window]   FIGURE 4. A, stereo view of the superposition of the active sites of StHK in complex with glucose (green) and human GlcNAc kinase in complex with GlcNAc (dark blue) based on the large domains. Hydrogen bonds between ligands and the small domains are shown as dashed lines. The residues from the adjacent subunits are indicated by primes. B, stereo view of the superposition of the overall structures of StHK in complex with glucose (green) and human GlcNAc kinase in complex with GlcNAc (dark blue) based on the large domains. C, stereo view of the superposition of the active sites of StHK in complex with glucose (green) and human hexokinase in complex with glucose and ADP (Protein Data Bank code 1DGK; magenta) based on the positions of glucose. The bound ADP molecule is not shown for clarity.

View larger version (31K): [in this window] [in a new window]   FIGURE 5. Modeling of glucose and ATP in the active site of the xylose·Mg2+·ADP complex. Modeled glucose and ATP are shown in stick representation with carbon atoms in yellow. The Mg2+ ion is shown as a green sphere. Water molecules coordinating the Mg2+ ion are shown as red spheres. WatA and WatB are shown as gray spheres. Hydrogen bonds between the enzyme and the water molecules coordinating the Mg2+ ion and between the enzyme and the -phosphate of ATP are indicated by dashed lines.

The crystal structures presented here confirm the catalytic mechanism proposed for the hexokinase family. Modeling of glucose and ATP in the active site of the xylose·Mg²⁺·ADP complex based on the positions of xylose and ADP shows that the 6-hydroxyl group of glucose is positioned at 2.8 Å from the -phosphorus of ATP (Fig. 5), which is suggestive of an in-line attack (55). In the glucose complex, O-1 of Asp-95 is 2.7 Å from the 6-hydroxyl group of glucose. We prepared the D95A mutant where Asp-95 is replaced by Ala. The mutation abolished the activity (less than 10^-5 compared with that of the wild-type enzyme), indicating the importance of Asp-95 for catalysis. In the active site of the xylose·Mg²⁺·ADP complex, a well ordered water molecule (Wat_A) is located at a distance of 2.6 Å from the -phosphoryl oxygen of ADP and forms a direct hydrogen bond with the backbone amides of Thr-116 and Gly-117 (Fig. 5). One of the -phosphoryl oxygens of the modeled ATP occupies a position similar to that of Wat_A to interact with Thr-116 and Gly-117, whereas another oxygen atom of the -phosphoryl can be placed within hydrogen bonding distance (2.6 Å) of the backbone amide of Gly-11 (Fig. 5). These observations indicate that the 1-2 and 6-7 turns are important for binding of both the - and -phosphates of ATP, which is consistent with the previously proposed models based on the crystal structures of human hexokinase I (17) and human GlcNAc kinase (39). The other -phosphoryl oxygen is positioned in close proximity to the water molecules coordinating the Mg²⁺ ion (Wat_B), suggesting that during catalysis this -phosphoryl oxygen might coordinate the ion in place of Wat_B with the - and -phosphates bridged by the ion. The Mg²⁺ ion together with Lys-15 would play a crucial role in stabilizing the developing negative charge on the phosphoryl oxygens during the transition state to promote catalysis.

The catalytic mechanism where an aspartate residue abstracts the proton from the 6-hydroxyl group of glucose at the initial stage of the reaction is commonly proposed for the hexokinase family. However, an aspartate is not likely able to abstract the proton from the 6-hydroxyl group of glucose because the pK_a of an aspartate is low. It may be more likely that the phosphoryl transfer would proceed through the metaphosphate-like transition state and an aspartate residue would accept the substrate proton from at the later stage, as proposed for some other kinases (56). In both cases, an aspartate residue would play an essential role in accepting the proton from the 6-hydroxyl group of glucose during catalysis.

Biological Implications—Here we describe the structural basis of the specificity of StHK for both glucose and GlcNAc. It has been reported that several enzymes involved in sugar metabolism in Sulfolobus also exhibit broad substrate specificity. In Sulfolobus, glucose is metabolized to pyruvate via the nonphosphorylative Entner-Doudoroff pathway (31). In the pathway, glucose dehydrogenase catalyzes the oxidation of glucose to gluconate, and then gluconate dehydratase catalyzes the dehydration of gluconate to KDG. Subsequently, KDG aldolase catalyzes the cleavage of KDG to glyceraldehyde and pyruvate. These three enzymes are involved in the metabolism of not only glucose but also galactose (57–59). Crystal structures of glucose dehydrogenase (60) and KDG aldolase (61) have been solved, and the structural bases for their substrate promiscuity have been determined. In addition, the nucleotidyltransferase from S. tokodaii has been reported to exhibit both glucose-1-phosphate thymidylyltransferase and GlcNAc-1-phosphate uridylyltransferase activities (62). No genes encoding sugar kinases responsible for phosphorylation of mannose, glucosamine, and GlcNAc have been found in the genomes of Sulfolobus species, suggesting that StHK may also be involved in phosphorylation of these sugars in vivo. Promiscuous enzymes may be generally used in some metabolic pathways in addition to central metabolism in Sulfolobus, which is in contrast with the situation in most other organisms where separate enzymes with higher specificity are usually used.

	FOOTNOTES

 
The atomic coordinates and structure factors (code 2E2N, 2E2O, 2E2P, and 2E2Q) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists 10865 (to H. N.) and the National Project on Protein Structural and Functional Analysis. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1 and Refs. 63 and 64.

¹ To whom correspondence should be addressed. Tel.: 81-3-5841-5152; Fax: 81-3-5841-5152; E-mail: atwakag{at}mail.ecc.u-tokyo.jp.

² The abbreviations used are: ROK, repressors/open reading frames of unknown function/sugar kinases; StHK, S. tokodaii hexokinase; PEG, polyethylene glycol; r.m.s.d., root mean square deviation; KDG, 2-keto-3-deoxygluconate.

	ACKNOWLEDGMENTS

 
We thank the staff at the Photon Factory for the x-ray data collection.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 

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