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Originally published In Press as doi:10.1074/jbc.M600801200 on May 1, 2006

J. Biol. Chem., Vol. 281, Issue 28, 19740-19747, July 14, 2006
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Crystal Structures of N-Acetylglucosamine-phosphate Mutase, a Member of the {alpha}-D-Phosphohexomutase Superfamily, and Its Substrate and Product Complexes*

Yuichi Nishitani{ddagger}, Daisuke Maruyama{ddagger}, Tsuyoshi Nonaka{ddagger}, Akiko Kita{ddagger}, Takaaki A. Fukami§, Toshiyuki Mio§, Hisafumi Yamada-Okabe§, Toshiko Yamada-Okabe, and Kunio Miki{ddagger}||1

From the {ddagger}Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan, §Kamakura Research Laboratory, Chugai Pharmaceutical Company Ltd., 200 Kajiwara, Kamakura, Kanagawa, 247-8530 Japan, Department of Hygiene, School of Medicine, Yokohama City University, 3-9, Fukuura, Kanazawa, Yokohama 236-0004, Japan, and ||RIKEN SPring-8 Center at Harima Institute, Koto 1-1-1, Sayo, Hyogo 679-5148, Japan

Received for publication, January 26, 2006 , and in revised form, April 4, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
N-Acetylglucosamine-phosphate mutase (AGM1) is an essential enzyme in the synthetic process of UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is a UDP sugar that serves as a biosynthetic precursor of glycoproteins, mucopolysaccharides, and the cell wall of bacteria. Thus, a specific inhibitor of AGM1 from pathogenetic fungi could be a new candidate for an antifungal reagent that inhibits cell wall synthesis. AGM1 catalyzes the conversion of N-acetylglucosamine 6-phosphate (GlcNAc-6-P) into N-acetylglucosamine 1-phosphate (GlcNAc-1-P). This enzyme is a member of the {alpha}-D-phosphohexomutase superfamily, which catalyzes the intramolecular phosphoryl transfer of sugar substrates. Here we report the crystal structures of AGM1 from Candida albicans for the first time, both in the apoform and in the complex forms with the substrate and the product, and discuss its catalytic mechanism. The structure of AGM1 consists of four domains, of which three domains have essentially the same fold. The overall structure is similar to those of phosphohexomutases; however, there are two additional beta-strands in domain 4, and a circular permutation occurs in domain 1. The catalytic cleft is formed by four loops from each domain. The N-acetyl group of the substrate is recognized by Val-370 and Asn-389 in domain 3, from which the substrate specificity arises. By comparing the substrate and product complexes, it is suggested that the substrate rotates about 180° on the axis linking C-4 and the midpoint of the C-5—O-5 bond in the reaction.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
UDP-N-acetylglucosamine (UDP-GlcNAc) is a UDP sugar that is synthesized from fructose 6-phosphate (Fru-6-P) supplied from the glycolytic pathway (1-4). UDP-GlcNAc is an essential metabolite that serves as a biosynthetic precursor of glycoproteins, mucopolysaccharides, and the cell wall of bacteria. Recently, it has been found that aberrant modification of O-linked N-acetylglucosamine, which is synthesized from UDP-GlcNAc, is closely linked with diabetes, cancer, and Alzheimer disease (5). The biosynthetic mechanisms of UDP-GlcNAc in prokaryotes and eukaryotes are very similar, but there are significant differences between them (6) (Fig. 1). In eukaryotes, UDP-GlcNAc is synthesized from Fru-6-P by four successive reactions (1-4): (i) the conversion of Fru-6-P into glucosamine 6-phosphate (GlcN-6-P) by glutamine-Fru-6-P amidotransferase, (ii) the acetylation of GlcN-6-P into N-acetylglucosamine 6-phosphate (GlcNAc-6-P) by GlcN-phosphate acetyltransferase, (iii) the interconversion of GlcNAc-6-P and N-acetylglucosamine 1-phosphate (GlcNAc-1-P) by N-acetylglucosamine-phosphate mutase (AGM1),2 and (iv) the uridylation of GlcNAc-1-P into UDP-GlcNAc by UDP-GlcNAc pyrophosphorylase (UAP1). In prokaryotes, intramolecular phosphoryl transfer (step iii) occurs before acetylation (step ii). Especially in Escherichia coli, acetylation and subsequent uridylation are catalyzed by a bifunctional enzyme, GlmU (7, 8).

AGM1, an essential enzyme that reversibly catalyzes the conversion of GlcNAc-6-P into GlcNAc-1-P, is found only in eukaryotes because of the above described differences in the synthetic routes of UDP-GlcNAc. AGM1 requires Mg2+ ions to achieve its maximum activity, but the reaction is inhibited by Zn2+ ions (9, 10). This enzyme is a member of the {alpha}-D-phosphohexomutase superfamily, which catalyzes an intramolecular phosphoryl transfer on sugar substrates. Although several structures of enzymes in the superfamily have been reported (11-13), the structure of AGM1 has not been determined yet. It is essential to reveal the three-dimensional structure of this enzyme in order to understand its catalytic mechanism and other important differences among enzymes in this superfamily.

Candida albicans is an opportunistic pathogen that causes life-threatening systemic infection in immunocompromised hosts (so called "candidiasis") (14). It is impossible for fungi to live without biosynthesis of the cell wall, and the chitin of the cell wall is synthesized from UDP-GlcNAc. A specific inhibitor of AGM1 from pathogenetic fungi could be a new candidate for an antifungal reagent that inhibits cell wall synthesis. Such a reagent would be expected to have fewer side effects because it acts on the cell wall, which does not exist in humans. In this article, we report the crystal structures of AGM1 from C. albicans (CaAGM1; 544 residues, 60 kDa) for the first time, both in the apoform and in complexes with substrates Formula and products Formula, and discuss the catalytic mechanism. Although the reaction catalyzed by AGM1 is reversible, in this study we assigned GlcNAc-6-P and GlcNAc-1-P as the substrate and product, respectively, to avoid confusion.


Figure 1
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  		FIGURE 1.
Schematic illustration of the role of AGM1 in the biosynthetic pathway of UDP-GlcNAc. In eukaryotes, UDP-GlcNAc is synthesized from fructose 6-phosphate by four successive reactions: (i) the conversion of Fru-6-P into GlcN-6-P; (ii) the acetylation of GlcN-6-P into GlcNAc-6-P; (iii) the interconversion of GlcNAc-6-P and GlcNAc-1-P; and (iv) the uridylation of GlcNAc-1-P into UDP-GlcNAc. AGM1 is catalyzed in step iii.In prokaryotes, the intramolecular phosphoryl transfer (step iii) occurs before acetylation (step ii), and GlcN-1-P is generated as the intermediate.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Purification, Crystallization, and Data Collection—CaAGM1 was overproduced, purified, and crystallized as described previously (6, 15). CaAGM1 was expressed as a glutathione S-transferase fusion and purified with a glutathione-Sepharose 4 FastFlow column (Amersham Biosciences) and a DEAE-Toyo-pearl 650 column (Tosoh, Tokyo). Crystals were obtained within a week using a reservoir solution (pH 4.6) containing 200 mM NH4H2PO4 and 14-20% (w/v) polyethylene glycol 3,350. Two different types of cryoprotectant solution were prepared. Cryoprotectant 1 contained the reservoir solution and 15% (v/v) glycerol, and cryoprotectant 2, which was used for the preparation of Hg derivatives to avoid precipitation, contained 200 mM acetate buffer (pH 4.5), 20% (w/v) polyethylene glycol 3,350, and 15% (v/v) glycerol. Pt derivatives were prepared by soaking in cryoprotectant 1 with 10 mM K2PtCl4 for 7 min. Hg derivatives were prepared by soaking in cryoprotectant 2 with 50% saturated p-chloromercury benzoate for 1 day. The substrate and product complexes were prepared by soaking in cryoprotectant 1 with each ligand (25 mM) and 5mM ZnCl2 for 1 min. All crystals were cryocooled in an N2 gas stream at 95 K. Diffraction data were collected using synchrotron radiation at SPring-8 (BL44B2 with a Mar CCD detector) and at the Photon Factory (BL5 with an ADSC Quantum 315 CCD detector and BL6A with an ADSC Quantum 4R CCD detector) and also using Cu-K{alpha} radiation from a rotating anode generator with RAXIS-IV and RAXIS-VII imaging plate detectors. All data sets were processed and scaled using the programs DENZO and SCALEPACK from the HKL2000 package (16).


Figure 2
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  		FIGURE 2.
Overall structures of CaAGM1. A and B, front view (A) and top view (B) of the ribbon diagram. Domains 1-4 are colored in green, yellow, red, and blue, respectively. A GlcNAc-1-P molecule and a Formula ion are shown as ball- and-stick models, where carbon, oxygen, nitrogen, and phosphorus atoms are presented in black, red, blue, and magenta, respectively. A Zn2+ ion is shown as a purple sphere. C, topology diagram of CaAGM1 with the same coloring scheme as in A. Helices are indicated by rectangles and beta-strands by arrows. The active-site regions are marked with asterisks. In domains 1-3, the four common strands are numbered, and helices and strands that are not part of the common structure are shown in lighter colors.

 
Phasing, Model Building, and Refinement—The crystal structure of the apoenzyme was solved by the multiple isomorphous replacement method using the programs SOLVE (17) and RESOLVE (18). The structural model was constructed using the program O (19). The crystal structures of the substrate and product complexes were determined by the molecular replacement method using the apoenzyme structure as a search model by the program MOLREP (20) in CCP4i (21, 22). All structures were refined using the program CNS (crystallography NMR software) (23). The atomic coordinates of CaAGM1 have been deposited in the Protein Data Bank under accession numbers 2DKA (apoenzyme), 2DKC (substrate complex), and 2DKD (product complex). The accessible surface area was calculated using the program SURFACE (24, 25) in CCP4 (21). Structures were superimposed using the program LSQMAN (26). Figures of structures were prepared with the program MOLSCRIPT (27) and Raster3D (28).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Overall Structure of AGM1—The crystal structure of CaAGM1 was determined at 1.93 Å resolution. Final statistics for all data sets are shown in Table 1. In all structures, approximately 90% of the residues are in the most favored region of the Ramachandran plot defined by PROCHECK (22, 29). It is suggested that the two molecules in the asymmetric unit of the crystal, which are related by noncrystallographic 2-fold symmetry, do not form a biological dimer, because each molecule of this pair buries only 4% of its total solvent-accessible surface in intermolecular contacts, an area outside the range expected for oligomeric proteins (30). Gel-filtration chromatography analysis (data not shown) also supported this hypothesis.


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  		TABLE 1
Crystallographic statistics

 
AGM1 contains domains 1 (residues 1-191), 2 (residues 192-311), 3 (residues 312-456), and 4 (residues 457-544). They are arranged in a "heart shape" (Fig. 2, A and B). Domains 1-3 fold similarly, with four beta-strands located between two {alpha}-helices (Fig. 2C). Domain 4 has a completely different fold, which contains two antiparallel beta-sheets. It has less interaction with the other domains and thus is more mobile. In the electron density map of the apoenzyme, domain 4 in one of two noncrystallographic symmetric molecules is disordered. However, in the structures of the substrate and product complexes, the electron density of domain 4 is clearly observed because of the binding of the substrate.


Figure 3
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  		FIGURE 3.
Structure-based amino acid sequence alignment between CaAGM1 and PaPMM/PGM. Their secondary structures are shown above and below the sequence alignment, respectively. Helices are indicated by rectangles and beta-strands by arrows. Identical residues are indicated by white letters on a red background, and similar residues are indicated by red letters. Active-site loops are outlined in black. PaPMM/PGM shares 20.7% sequence identity with CaAGM1.

 
Comparison with Phosphomannomutase/Phosphoglucomutase (PMM/PGM)—PMM/PGM is also a member of the {alpha}-D-phosphohexomutase superfamily and catalyzes the reversible conversion of mannose 6-phosphate into mannose 1-phosphate and glucose 6-phosphate into glucose 1-phosphate (12, 13). PMM/PGM from Pseudomonas aeruginosa (PaPMM/PGM) has 463 residues and shares 20.7% sequence identity with CaAGM1 (Fig. 3) as calculated by the program FASTA (31, 32).

The superposition of CaAGM1 (the product complex) with PaPMM/PGM (the complex with mannose 1-phosphate; Protein Data Bank code 1PCJ) is shown in Fig. 4A. These two structures, which are essentially identical, are composed of four domains arranged in a heart shape. In particular, the activesite residues are well superimposed (Fig. 4B). For AGM1, domain 4 has two additional beta-strands (the red region in Fig. 4A), which were not observed in the structures of the superfamily. These strands are superimposed with an {alpha}-helix in PMM/PGM.


Figure 4
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  		FIGURE 4.
Comparison between AGM1 and PMM/PGM. A, front view of the superposition of AGM1 with PMM/PGM. C{alpha} traces of AGM1 and PMM/PGM are shown in cyan and yellow, respectively. Two additional beta-strands are shown in red. B, superposition of the active-site residues. C, circular permutation in domain 1. The topology diagram is drawn as described for Fig. 2C. First, the core structure of PMM/PGM is selected. Next, the N and C termini are fused. The bold red line is cleaved to form the new N and C termini. As a result, the structure obtained is the same as the core structure of AGM1.

 


Figure 5
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  		FIGURE 5.
Superposition of the three structures of AGM1. A, superposition of the overall structures of AGM1. C{alpha} traces of the apoform, the substrate complex, and the product complex are shown in green, red, and blue, respectively. The flexible active-site loop observed only in the substrate and product complexes is shown by a red dot circle. GlcNAc-6-P (the substrate) and GlcNAc-1-P (the product) are shown in magenta and white, respectively. Formula and Zn2+ ions are shown as described for Fig. 2A. B, a macrograph about the substrate and the product. The N-acetyl groups are indicated by blue circles. The axis of rotation is indicated by the green line.

 
Circular Permutation—For AGM1 and PMM/PGM, domains 1-3 are structurally similar to each other, each having four beta-strands located between two {alpha}-helices. The topology diagrams for domains 2 and 3 are essentially the same between these proteins. The beta-strands are arranged in the order of 2-1-3-4, and strand 4 is antiparallel to the other three strands. However, the diagram for domain 1 is completely different, as shown in Fig. 4C. In this domain, the strands are arranged in different order. For AGM1, the beta-strands are arranged in the order of 4-3-1-2, and strand 2 is antiparallel to the other three strands. For all of the other proteins in the superfamily, the order is the same as in PMM/PGM (11). The active-site residues are well superimposed, and the sequences around the active-site residues are conserved between AGM1 and PMM/PGM (Fig. 3). It is known that circular permutation can occur by the spatial adjacency of the N and C termini of the polypeptide chain (33, 34). This mutation involves the fusion of the N and C termini and cleavage at another site. Such a mutation is observed in domain 1 of this enzyme (Fig. 4C). The distance on the amino acid sequence between the active-site residues of domains 1 and 2 is 224 residues in AGM1, whereas it is only 134 residues in PMM/PGM. This large gap suggests that AGM1 evolutionarily diverged from the superfamily in the earliest period (35). Although it has been suggested previously that an insert sequence probably exists (36), our structural analysis demonstrates that the difference lies in the strand order.


Figure 6
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  		FIGURE 6.
Stereoview of the environment around the active site of AGM1. The substrate (A) and product complexes (B) are shown. Colored loops are the active serine loop (I), the metal-binding loop (II), the sugarbinding loop (III), and the phosphate-binding loop (IV). Domains and atoms are shown in the same colors as described for Fig. 2 (except for carbon atoms). The electron density, shown in cyan, is contoured at 3.0 {sigma} with the program CONSCRIPT (37).

 
Active Site—The crystal structures of the complexes with substrates Formula and products Formula were also determined at 2.20 and 2.10 Å resolution, respectively (Fig. 5A). The Formula ion was found in these complexes but not in the apoform. The substrate (GlcNAc-6-P) and product (GlcNAc-1-P) molecules were observed in the active site. The position of the active site in this enzyme is similar to those found in other enzymes belonging to the {alpha}-D-phosphohexomutase superfamily (35, 36). The catalytic cleft is formed by four loops from each domain (Fig. 6): the active serine loop (Thr-64-Glu-71 in domain 1), the metal-binding loop (Asp-290-Arg-295 in domain 2), the sugar-binding loop (Glu-387-His-391 in domain 3), and the phosphate-binding loop (Arg-512-Ala-519 in domain 4). The residues identified as affecting the activity by previous mutation analysis are located on all of these loops except the sugar-binding loop (6).

The active serine loop contains the catalytic residue Ser-66 (Fig. 7, A and B). This residue is observed as the phosphorylated form in the superfamily (11, 12). However, judging from the electron density maps, it is not phosphorylated in all structures of the apoform and the substrate and product complexes. In the substrate and product complexes, the O{gamma} atom of Ser-66 is not covalently bonded to the Formula ion. In the apoenzyme, Ser-66 is disordered in one of two noncrystallographic symmetric molecules. On the other hand, in the substrate and product complexes, it is clearly observed but is in the disallowed region of the Ramachandran plot defined by PROCHECK (22, 29) under the influence of the Zn2+ ion. The active serine loop is fixed by the Zn2+ ion through Ser-66.

The metal-binding loop plays an important role (Fig. 7, A and B). A metal ion is essential for the activity of AGM1. In the apoenzyme, no metal ion is observed at this position, and the side chains of three aspartic acid residues (Asp-290,-292, and -294) on this loop point to various positions. On the other hand, in the substrate and product complexes, the Zn2+ ion is chelated to the side chains of these residues. In both cases, the above features of the aspartic acid residues are clearly observed in the electron density maps. The charge of the metal ion becomes more positive in the transition state, and probably the Formula ion is smoothly transferred between the O{gamma} atom of Ser-66 and the O-6 or O-1 atom of the substrate (12).

The sugar-binding loop binds to the hydroxyl groups of the substrate (Fig. 7, C and D). In the substrate and product complexes, the side chain of Glu-387 is hydrogen-bonded to the O-3 and O-4 atoms of the sugar ring. In addition, one water molecule is hydrogen-bonded to the O-4 atom in the substrate complex, whereas the side chain of Asn-389 is hydrogen-bonded to the O-3 atom in the product complex. Compared with the substrate and product complexes, the sugar rings of the substrate (GlcNAc-6-P) and product (GlcNAc-1-P) molecules are related by a 180° rotation around an axis linking the C-4 atom and the midpoint of the C-5—O-5 bond (Fig. 5B). It is suggested that the substrate (the reaction intermediate) rotates 180° around the axis in the reaction.


Figure 7
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  		FIGURE 7.
Close-up view of AGM1. A and B, the active-site serine and the metal-binding loop in the apoenzyme (A) and the product complex (B). C and D, the sugar-binding loop in the substrate (C) and product (D) complexes. E and F, the phosphate-binding loop in the substrate (E) and product (F) complexes. G and H, the N-acetyl group in the substrate (G) and product (H) complexes. Each part of this figure is drawn in the same way as in Fig. 6. Water molecules are shown as blue spheres. Hydrogen bonds are indicated by dashed lines.

 


Figure 8
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  		FIGURE 8.
Schematic drawing of the proposed catalytic mechanism for the conversion of GlcNAc-6-P to GlcNAc-1-P by AGM1. Phosphoryl groups are indicated by P in a shaded circle. The phosphoryl group near Ser-66 first binds to the substrate. The substrate is converted into a bis-phosphorylated intermediate. Then, this intermediate rotates at 180°. Consequently, the phosphoryl group at C-6 changes positions with another phosphoryl group at C-1. Finally, the phosphoryl group moved near the metal ion dissociates from the intermediate and binds to Ser-66.

 
The phosphate-binding loop, located at the opposite side of the active serine loop, interacts with the phosphoryl group of the substrate (Fig. 7, E and F). This loop is particularly flexible, because in the apoenzyme this loop is not visible in the electron density map. In the substrate and product complexes, however, it is clearly observed (the red dot circle in Fig. 5A). It also plays the role of covering the active-site cleft. The phosphoryl group of the substrate is hydrogen-bonded to the side chains of Arg-512, Ser-514, Gly-515, Thr-516, and Arg-521. The binding modes in the substrate and product complexes are very similar to each other. In addition, two water molecules are hydrogen-bonded to the phosphoryl group in the substrate complex. But only one water molecule binds to the phosphoryl group in the product complex. The N-acetyl group of the substrate in the product complex is replaced by the water molecule in the substrate complex.

Substrate Specificity—The N-acetyl group of the substrate is responsible for the substrate specificity (Fig. 7, G and H). In the substrate complex, the carbonyl oxygen atom of the N-acetyl group is hydrogen-bonded to the main chain N atom of Lys-371 through a water molecule. In addition, the N-acetyl group is located near the side chain of Val-370 at a distance of 3.24 Å. A sufficiently large space is indispensable for the recognition of the substrate. If Val-370 were much larger, steric hindrance would occur. Val-370 of CaAGM1 is replaced by the histidine residue of PMM/PGM, the aspartic acid residue of phosphoglucosamine mutase, and the tryptophan residue of PGM. Phosphoglucosamine mutase and PGM are also members of the {alpha}-D-phosphohexomutase superfamily. It is difficult for such large residues of other proteins in the superfamily to accept the N-acetyl group. Consequently, it is suggested that a small side chain as the valine residue is suitable to recognize the N-acetyl group of the substrate.

On the other hand, in the product complex, the carbonyl oxygen atom of the N-acetyl group is hydrogen-bonded to a water molecule and the side chain of Asn-389. Interestingly, Asn-389 of CaAGM1 is replaced by the serine residues of all the other proteins in the superfamily (35, 36). It is assumed that this residue is important to the recognition of the N-acetyl group of the substrate.

Catalytic Mechanism—The catalytic mechanism of AGM1 can be understood in the same way as those proposed for other proteins in the superfamily (12, 13). It is considered that the catalytic mechanism requires two phosphoryl transfer reactions: the nucleophilic substitution on the phosphorus atom by the Sn2 mechanism and the cleavage of the phosphoanhydride bond (Fig. 8). First, the phosphoryl group from the phosphorylated Ser-66 binds to the substrate. In the present structure, Ser-66 is not phosphorylated. But it is assumed that this reaction is not done without the phosphorylation of this serine residue, because a hydroxyl group of a Formula ion by itself is too basic to be substituted for the hydroxyl group of the substrate at C-6 or C-1. The substrate is converted into a bis-phosphorylated intermediate. This hypothetical pathway via the intermediate could be supported by the measurement of the activity with sugar 1,6-diphosphates and the experiment detecting the intermediate (10). Then, this intermediate rotates 180°. Consequently, the phosphoryl group at C-6 changes positions with another phosphoryl group at C-1. Finally, the phosphoryl group, which has moved near the metal ion, dissociates from the intermediate and binds to Ser-66.

The active-site cleft is too narrow for this intermediate to rotate. The active-site cleft could be expanded by the movement of domain 4. The bis-phosphorylated intermediate is released from the active site and then rotates 180° and is accommodated again to the active-site cleft. In this event, the metal ion plays an important role. As seen in the apoenzyme, when a Zn2+ ion is absent, a Formula ion is also unobserved. This indicates that the metal ion is indispensable for the holding of the Formula ion. Furthermore, as mentioned above, the positive charge from the metal ion could contribute to the transfer of the Formula ion (38, 39).


   FOOTNOTES
 
The atomic coordinates and structure factors (codes 2DKA, 2DKC, 2DKD) 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 a grant of the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. 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. Back

1 To whom correspondence should be addressed: Dept. of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. Tel.: 81-75-753-4029; Fax: 81-75-753-4032; E-mail: miki{at}kuchem.kyoto-u.ac.jp.

2 The abbreviations used are: AGM1, N-acetylglucosamine-phosphate mutase; CaAGM1, AGM1 from Candida albicans; PMM/PGM, phosphomannomutase/phosphoglucomutase; PaPMM/PGM, PMM/PGM from Pseudomonas aeruginosa; CCD, charge-coupled device. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Satoshi Sogabe for the biochemical experiments and to the beamline scientists at SPring-8 and the Photon Factory.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

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