Collective motions in glucosamine-6-phosphate synthase: influence of ligand binding and role in ammonia channelling and opening of the fructose-6-phosphate binding site

The large protein motions of the bacterial enzyme glucosamine-6-phosphate synthase have been addressed using full atom normal modes analysis for the empty, the glucose-6-phosphate and the glucose-6-phosphate + glutamate bound proteins. The approach that was used involving energy minimizations along the normal modes coordinates identified functional motions of the protein, some of which were characterized earlier by X-ray diffraction studies. This method made it possible for the first time to highlight significant energy differences according to whether none, only one or both of the active sites of the protein were occupied. Our data favoured a specific motion of the glutamine binding domain following the fixation of fructose-6-phosphate and suggested a rigidified structure with both sites occupied. Here, we show that most of the collective large amplitude motions of glucosamine-6-phosphate synthase that are modulated by ligand binding are crucial for the enzyme catalytic cycle, as they strongly modify the geometry of both the ammonia channel and the C-tail, demonstrating their role in ammonia transfer and ligand binding.     

Glucosamine-6-phosphate synthase, a novel target for antifungal agents. Molecular modelling studies in drug design

Fungal infections are a growing problem in contemporary medicine, yet only a few antifungal agents are used in clinical practice. In our laboratory we proposed the enzyme L-glutamine: D-fructose-6-phosphate amidotransferase (EC 2.6.1.16) as a new target for antifungals. The structure of this enzyme consists of two domains, N-terminal and C-terminal ones, catalysing glutamine hydrolysis and sugar-phosphate isomerisation, respectively. In our laboratory a series of potent selective inhibitors of GlcN-6-P synthase have been designed and synthesised. One group of these compounds, including the most studied N3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid (FMDP), behave like glutamine analogs acting as active-site-directed inactivators, blocking the N-terminal, glutamine-binding domain of the enzyme. The second group of GlcN-6-P synthase inhibitors mimic the transition state of the reaction taking place in the C-terminal sugar isomerising domain. Surprisingly, in spite of the fact that glutamine is the source of nitrogen for a number of enzymes it turned out that the glutamine analogue FMDP and its derivatives are selective against GlcN-6-P synthase and they do not block other enzymes, even belonging to the same family of glutamine amidotransferases. Our molecular modelling studies of this phenomenon revealed that even within the family of related enzymes substantial differences may exist in the geometry of the active site. In the case of the glutamine amidotransferase family the glutamine binding site of GlcN-6-P synthase fits a different region of the glutamine conformational space than other amidotransferases. Detailed analysis of the interaction pattern for the best known, so far, inhibitor of the sugar isomerising domain, namely 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP), allowed us to suggest changes in the structure of the inhibitor that should improve the interaction pattern. The novel ligand was designed and synthesised. Biological experiments confirmed our predictions. The new compound named ADMP is a much better inhibitor of glucosamine-6-phosphate synthase than ADGP.     

Glutamine binding opens the ammonia channel and activates glucosamine-6P synthase

Glucosamine-6P synthase catalyzes the synthesis of glucosamine-6P from fructose-6P and glutamine and uses a channel to transfer ammonia from its glutaminase to its synthase active site. X-ray structures of glucosamine-6P synthase have been determined at 2.05 Angstroms resolution in the presence of fructose-6P and at 2.35 Angstroms resolution in the presence of fructose-6P and 6-diazo-5-oxo-L-norleucine, a glutamine affinity analog that covalently modifies the N-terminal catalytic cysteine, therefore mimicking the gamma-glutamyl-thioester intermediate formed during hydrolysis of glutamine. The fixation of the glutamine analog activates the enzyme through several major structural changes: 1) the closure of a loop to shield the glutaminase site accompanied by significant domain hinging, 2) the activation of catalytic residues involved in glutamine hydrolysis, i.e. the alpha-amino group of Cys-1 and Asn-98 that is positioned to form the oxyanion hole, and 3) a 75 degrees rotation of the Trp-74 indole group that opens the ammonia channel.     

Structural Basis for Morpheein-type Allosteric Regulation of Escherichia coli Glucosamine-6-phosphate Synthase: EQUILIBRIUM BETWEEN INACTIVE HEXAMER AND ACTIVE DIMER*

The amino-terminal cysteine of glucosamine-6-phosphate synthase (GlmS) acts as a nucleophile to release and transfer ammonia from glutamine to fructose 6-phosphate through a channel. The crystal structure of the C1A mutant of Escherichia coli GlmS, solved at 2.5 Å resolution, is organized as a hexamer, where the glutaminase domains adopt an inactive conformation. Although the wild-type enzyme is active as a dimer, size exclusion chromatography, dynamic and quasi-elastic light scattering, native polyacrylamide gel electrophoresis, and ultracentrifugation data show that the dimer is in equilibrium with a hexameric state, in vitro and in cellulo. The previously determined structures of the wild-type enzyme, alone or in complex with glucosamine 6-phosphate, are also consistent with a hexameric assembly that is catalytically inactive because the ammonia channel is not formed. The shift of the equilibrium toward the hexameric form in the presence of cyclic glucosamine 6-phosphate, together with the decrease of the specific activity with increasing enzyme concentration, strongly supports product inhibition through hexamer stabilization. Altogether, our data allow us to propose a morpheein model, in which the active dimer can rearrange into a transiently stable form, which has the propensity to form an inactive hexamer. This would account for a physiologically relevant allosteric regulation of E. coli GlmS. Finally, in addition to cyclic glucose 6-phosphate bound at the active site, the hexameric organization of E. coli GlmS enables the binding of another linear sugar molecule. Targeting this sugar-binding site to stabilize the inactive hexameric state is therefore suggested for the development of specific antibacterial inhibitors.     

Functional domains and interdomain communication in Candida albicans glucosamine-6-phosphate synthase

Functional and structural properties of several truncated or mutated variants of Candida albicans Gfa1p (glucosamine-6-phosphate synthase) were compared with those of the wild-type enzyme. Fragments encompassing residues 1-345 and 346-712 of Gfa1p, expressed heterogeneously in bacterial host as His6 fusions, were identified as the functional GAH (glutamine amidehydrolysing) and ISOM (hexose phosphate-isomerizing) domains respectively. It was found that the native GAH domain is monomeric, whereas the native ISOM domain forms tetramers, as does the whole enzyme. Spectrofluorimetric and kinetic studies of the isolated domains, the Delta218-283Gfa1p mutein and the wild-type enzyme revealed that the binding site for the feedback inhibitor, uridine 5'-diphospho-N-acetyl-D-glucosamine, is located in the ISOM domain. Inhibitor binding affects amidohydrolysing activity of the GAH domain and, as a consequence, the GlcN-6-P (D-glucosamine-6-phosphate)-synthetic activity of the whole enzyme. The fragment containing residues 218-283 is neither involved in ligand binding nor in protein oligomerization. Comparison of the catalytic activities of Gfa1p(V711F), Delta709-712Gfa1p, Gfa1p(W97F) and Gfa1p(W97G) with those of the native Gfa1p and the isolated domains provided evidence for an intramolecular channel connecting the GAH and ISOM domains of Gfa1p. The channel becomes leaky upon deletion of amino acids 709-712 and in the W97F and W97G mutants. The Trp97 residue was found to function as a molecular gate, opening and closing the channel. The W97G and V711F mutations resulted in an almost complete elimination of the GlcN-6-P-synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities.     

Domain motions of glucosamine-6P synthase: comparison of the anisotropic displacements in the crystals and the catalytic hinge-bending rotation

Glucosamine-6-phosphate synthase channels ammonia over 18 A from glutamine at the glutaminase site to fructose-6P at the synthase site. We have modeled the anisotropic displacements of the glutaminase and synthase domains from the two crystallized states, the enzyme in complex with fructose-6P or in complex with glucose-6P and a glutamine affinity analog, using TLS (rigid-body motion in terms of translation, libration, and screw motions) refinement implemented in REFMAC. The domains displacements in the crystal lattices are compared to the movement of the glutaminase domain relative to the synthase domain that occurs during the catalytic cycle upon glutamine binding, which was visualized by comparing the two structures. This movement was analyzed by the program DYNDOM as a 22.8 degrees rotation around an effective hinge axis running approximately parallel to helix 300-317 of the synthase domain, the glutaminase loop that covers the glutaminase site upon glutamine binding acting as the mechanical hinge.     

The high resolution crystal structure of yeast hexokinase PII with the correct primary sequence provides new insights into its mechanism of action

Hexokinase is the first enzyme in the glycolytic pathway, catalyzing the transfer of a phosphoryl group from ATP to glucose to form glucose 6-phosphate and ADP. Two yeast hexokinase isozymes are known, namely PI and PII. The crystal structure of yeast hexokinase PII from Saccharomyces cerevisiae without substrate or competitive inhibitor is determined and refined in a tetragonal crystal form at 2.2-A resolution. The folding of the peptide chain is very similar to that of Schistosoma mansoni and previous yeast hexokinase models despite only 30% sequence identity between them. Distinct differences in conformation are found that account for the absence of glucose in the binding site. Comparison of the current model with S. mansoni and yeast hexokinase PI structures both complexed with glucose shows in atomic detail the rigid body domain closure and specific loop movements as glucose binds. A hydrophobic channel formed by strictly conserved hydrophobic residues in the small domain of the hexokinase is identified. The channel's mouth is close to the active site and passes through the small domain to its surface. The possible role of the observed channel in proton transfer is discussed.     

Purification and characterization of glutamine:fructose 6-phosphate amidotransferase from rat liver

The enzyme glutamine:fructose 6-phosphate amidotransferase (L-glutamine:D-fructose-6-phosphate amidotransferase; EC 2.6.1.16, GFAT) catalyzes the formation of glucosamine 6-phosphate from fructose 6-phosphate and glutamine. In view of the important role of GFAT in the hexosamine biosynthetic pathway, we have purified the enzyme from rat liver and characterized its physicochemical properties in comparison to those from the published microbial enzymes. The purified enzyme has a molecular mass of about 75 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. On a Sephacryl S-200 gel filtration column, the purified enzyme eluted in a single peak corresponding to a molecular mass of about 280 kDa, indicating that the active enzyme may be composed of four subunits. The N-terminal amino acid sequence of the purified enzyme was determined as X-G-I-F-A-Y-L-N-Y-H-X-P-R, where X indicates an unidentified residue. The K(M) values of the purified enzyme for fructose 6-phosphate and glutamine were 0.4 and 0.8 mM, respectively. The purified enzyme was inactivated by 4, 4'-dithiodipyridine, and the activity of the inactivated enzyme was restored by dithiothreitol. The inactivation followed pseudo first-order and saturation kinetics with the K(inact) of 5.0 microM. Kinetic studies also indicated that 4,4'-dithiodipyridine is a competitive inhibitor of the enzyme with respect to glutamine. Isolation and analysis of the cysteine-modified peptide indicated that Cys-1 was the modified site. Cys-1 has been suggested to play an important role in enzymatic activity of the Escherichia coli enzyme (M. N. Isupov, G. Obmolova, S. Butterworth, M. Badet-Denisot, B. Badet, I. Polikarpov, J. A. Littlechild, and A. Teplyakov, 1996, Structure 4, 801-810).     

Ordering of C-terminal loop and glutaminase domains of glucosamine-6-phosphate synthase promotes sugar ring opening and formation of the ammonia channel

Glucosamine-6-phosphate synthase (GlmS) channels ammonia from glutamine at the glutaminase site to fructose 6-phosphate (Fru6P) at the synthase site. Escherichia coli GlmS is composed of two C-terminal synthase domains that form the dimer interface and two N-terminal glutaminase domains at its periphery. We report the crystal structures of GlmS alone and in complex with the glucosamine-6-phosphate product at 2.95 Å and 2.9 Å resolution, respectively. Surprisingly, although the whole protein is present in this crystal form, no electron density for the glutaminase domain was observed, indicating its mobility. Comparison of the two structures with that of the previously reported GlmS-Fru6P complex shows that, upon sugar binding, the C-terminal loop, which forms the major part of the channel walls, becomes ordered and covers the synthase site. The ordering of the glutaminase domains likely follows Fru6P binding by the anchoring of Trp74, which acts as the gate of the channel, on the closed C-terminal loop. This is accompanied by a major conformational change of the side chain of Lys503^# of the neighboring synthase domain that strengthens the interactions of the synthase domain with the C-terminal loop and completely shields the synthase site. The concomitant conformational change of the Lys503^#-Gly505^# tripeptide places catalytic His504^# in the proper position to open the sugar and buries the linear sugar, which is now in the vicinity of the catalytic groups involved in the sugar isomerization reaction. Together with the previously reported structures of GlmS in complex with Fru6P or glucose 6-phosphate and a glutamine analogue, the new structures reveal the structural changes occurring during the whole catalytic cycle.     

Inactivation of glucosamine-6-phosphate synthetase from Salmonella typhimurium LT2 by fumaroyl diaminopropanoic acid derivatives, a novel group of glutamine analogs

A novel group of glutamine analogs, N3-fumaroyl-L-2,3-diaminopropanoic acid (FDP) and its derivatives and analogs including amide (FCDP), methyl ester (FMDP) and its homologue, N4-(4-methoxyfumaroyl)-L-2,4-diaminobutanoic acid, inactivate glucosamine-6-phosphate synthetase (L-glutamine: D-fructose-6-phosphate aminotransferase (hexose-isomerizing), EC 2.6.1.16), isolated from Salmonella typhimurium, by covalent modification. For comparative purposes, selected known glutamine analogs were also examined. Anticapsin, 6-diazo-5-oxo-L-norleucine and, at high concentration, azaserine inactivate the enzyme. The pseudo-first-order rate constants show a hyperbolic dependence on inhibitor concentration for all the above-mentioned inhibitors, suggesting the formation of a reversible complex prior to covalent modification. Dissociation constants for inhibitors were determined and ranged from 10(-4) M for FCDP to 10(-6) M for FMDP. Albizziin, gamma-glutamylhydroxamate and, at low concentration, azaserine inhibit glucosamine synthetase only reversibly. All inhibitors tested are competitive in relation to glutamine. and competitive inhibitors, albizziin and gamma-glutamylhydroxamate protect the enzyme against inactivation. Fructose 6-phosphate accelerates the rate of inactivation. Some analogs of FDP, such as SMDP, CRDP, O-FMSer, MMDP and AADP, are not active against glucosamine-6-phosphate synthetase. The structure-activity relationship of the novel group of glutamine analogs is discussed and structural requirements for the activity of these compounds is established. It is postulated that the compounds examined can be classified as mechanism-based enzyme inactivators.     

A cysteine-histidine-aspartate catalytic triad is involved in glutamine amide transfer function in purF-type glutamine amidotransferases

A family of four glutamine amidotransferases has a homologous glutamine amide transfer domain, designated purF-type, that is named after purF-encoded glutamine phosphoribosylpyrophosphate amidotransferase. The glutamine amide transfer domain of approximately 194 amino acid residues is at the NH2 terminus of the protein chain. Site-directed mutagenesis was used to replace several of the 9 invariant amino acids in the glutamine amide transfer domain of glutamine phosphoribosylpyrophosphate amidotransferase. The results indicate that a Cys1-His101-Asp29 catalytic triad is involved in the glutamine amide transfer function of this enzyme. The evidence suggests that His101 functions to increase the nucleophilicity of Cys1, which is used to form a glutamine-enzyme covalent intermediate. Asp29 has a role subsequent to formation of the covalent intermediate. The Cys-His-Asp catalytic triad is implicated in the glutamine amide transfer function of purF-type amidotransferases.     

Glucosamine-6-phosphate synthase from Escherichia coli yields two proteins upon limited proteolysis: identification of the glutamine amidohydrolase and 2R ketose/aldose isomerase-bearing domains based on their biochemical properties

The proteolysis of native glucosamine-6-phosphate synthase (Mr 67,000) from Escherichia coli was investigated using two nonspecific and five specific endoproteinases, alpha-chymotrypsin generated two nonoverlapping polypeptides CT1 and CT2 of Mr 40,000 and 27,000 lacking glucosamine-6P synthesizing activity. Amino terminal and carboxy terminal sequence analysis showed that cleavage occurred between positions 240 and 241 of the primary sequence without further degradation. The glutamine amidohydrolase activity was located in the CT2 N-terminal polypeptide which was capable of incorporating 0.7 equivalent of the glutamine site-directed affinity label [2-3H]-N3-(4-methoxyfumaroyl)-diaminopropionic acid indicating that it bears the amidotransferase function. CT1 which displayed a higher reactivity than CT2 for fructose-6P binding contains the ketose/aldose isomerase activity. These data suggest the existence of a hinge structure essential for the catalytically efficient coupling between the ammonia generating domain and the sugar binding domain and support the model recently proposed by Mei and Zalkin in which purF-type amidotransferases contain a glutamine hydrolase domain of approximately 200 amino acids fused to an ammonia-transfer domain.     

Chemical modification of glucosamine-6-phosphate synthase by diethyl pyrocarbonate: evidence of histidine requirement for enzymatic activity.

Glucosamine-6-phosphate synthase from Escherichia coli was inactivated by diethylpyrocarbonate at pH 7.3 and 4 degrees C with a second-order rate constant of 1220 M-1 min-1. The difference spectrum of inactivated vs native enzyme had a maximum absorption at 242 nm, which is characteristic of N-carbethoxyhistidine. No trough at around 280 nm due to O-carbethoxytyrosine was observed and the sulfhydryl content of the enzyme was unchanged. Studies with [14C]diethylpyrocarbonate provided evidence that derivatization of a single histidine residue of the amino-terminal glutamine-binding domain inactivated glucosamine-6P synthase. These results are consistent with the participation of an histidine residue in a catalytic triad, Cys/His/Asp, necessary to generate ammonia from glutamine.     

Crystal structures of N-acetylglucosamine-phosphate mutase, a member of the alpha-D-phosphohexomutase superfamily, and its substrate and product complexes

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 degrees on the axis linking C-4 and the midpoint of the C-5-O-5 bond in the reaction.     

Crystal structure of pyruvate kinase from Geobacillus stearothermophilus

The pyruvate kinase (PK) from a moderate thermophile, Geobacillus stearothermophilus, is an allosteric enzyme activated by AMP and ribose 5-phosphate but not fructose 1, 6-bisphosphate (FBP), which is a common activator of PKs. It has an extra C-terminal sequence (ECTS), which contains a highly conserved phosphoenolpyruvate (PEP) binding motif, but its function and structure remain unclear. To elucidate the structural characteristics of the effector-binding site and the ECTS, the crystal structure of the C9S/C268S mutant of the enzyme was determined at 2.4 A resolution. The crystal belonged to space group P6(2)22, with unit cell parameters a, b = 145.97 A, c = 118.03 A. The enzyme was a homotetramer and its overall domain structure was similar to the previously solved structures except that the ECTS formed a new domain (C' domain). The structure of the C' domain closely resembled that of the PEP binding domain of maize pyruvate phosphate dikinase. A sulphate ion was found in a pocket in the effector-binding C domain. This site corresponds to the 6-phosphate group-binding site in yeast PK bound FBP and seems to be the effector-binding site. Through comparison of the structure of the putative effector-binding site to that of the FBP binding site of the yeast enzyme, the structural basis of the effector specificity of the G. stearothermophilus PK is discussed.     

The binding of substrates and modifiers to glucosamine synthetase

1. The binding of substrates and effectors to glucosamine synthetase (l-glutamine-d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) was studied by using the ligand to alter the denaturation rate of the enzyme. The free enzyme bound fructose 6-phosphate, glucose 6-phosphate and UDP-N-acetylglucosamine, but not glutamine, AMP or UTP. Glucose 6-phosphate and AMP increased the binding of UDP-N-acetylglucosamine whereas UTP decreased the interaction between the enzyme and the feedback inhibitor. UDP-N-acetylglucosamine induced a glutamine-binding site on the enzyme. 2. Selective thermal or chemical denaturation revealed that the UDP-N-acetylglucosamine-binding site was not located at the catalytic site. The UTP site could not be distinguished from that for the nucleotide sugar. The AMP- and glucose 6-phosphate-binding sites were distinct from the catalytic and feedback-inhibitor-binding sites. 3. The specificity of the glutamine-binding site was investigated by using a series of potential analogues. 4. A model is proposed for the action of the effectors and the mechanism of the reaction discussed in kinetic and chemical terms.     

Ammonia channeling in bacterial glucosamine-6-phosphate synthase (Glms): molecular dynamics simulations and kinetic studies of protein mutants

Ammonia transfer from the glutamine site to the fructose-6P site of bacterial glucosamine-6-phosphate synthase was studied by molecular dynamics simulations. The studies suggest a key role for Trp74, in the sealing of the hydrophobic channel connecting the two binding sites, as well as for the two Ala602 and Val605 residues, which form a narrow passage whose opening/closing constitutes an essential event in ammonia transfer. Kinetic analyses of the corresponding protein mutants confirmed our predictions. The efficiency of ammonia transfer which was close to zero in the W74A mutant was partially restored by increasing the size of the corresponding side-chain; the simulations performed on the W74A mutant suggested the formation of a hole in the channel. In the case of A602L and V605L mutants, the efficiency of ammonia transfer decreased to approximately 50% of the value of the native protein. None of the mutants were, however, able to use exogenous ammonia as a substrate.     

Glucosamine synthetase from Escherichia coli: purification, properties, and glutamine-utilizing site location

L-Glutamine:D-fructose-6-phosphate amidotransferase (glucosamine synthetase) has been purified to homogeneity from Escherichia coli. A subunit molecular weight of 70,800 was estimated by gel electrophoresis in sodium dodecyl sulfate. Pure glucosamine synthetase did not exhibit detectable NH3-dependent activity and did not catalyze the reverse reaction, as reported for more impure preparations [Gosh, S., Blumenthal, H. J., Davidson, E., & Roseman, S. (1960) J. Biol. Chem. 235, 1265]. The enzyme has a Km of 2 mM for fructose 6-phosphate, a Km of 0.4 mM for glutamine, and a turnover number of 1140 min-1. The amino-terminal sequence confirmed the identification of residues 2-26 of the translated E. coli glmS sequence [Walker, J. E., Gay, J., Saraste, M., & Eberle, N. (1984) Biochem. J. 224, 799]. Methionine-1 is therefore removed by processing in vivo, leaving cysteine as the NH2-terminal residue. The enzyme was inactivated by the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) and by iodoacetamide. Glucosamine synthetase exhibited half-of-the-sites reactivity when incubated with DON in the absence of fructose 6-phosphate. In its presence, inactivation with [6-14C]DON was accompanied by incorporation of 1 equiv of inhibitor per enzyme subunit. From this behavior, a dimeric structure was tentatively assigned to the native enzyme. The site of reaction with DON was the NH2-terminal cysteine residue as shown by Edman degradation.     

Crystal structure of MJ1247 protein from M. jannaschii at 2.0 A resolution infers a molecular function of 3-hexulose-6-phosphate isomerase

The crystal structure of the hypothetical protein MJ1247 from Methanococccus jannaschii at 2 A resolution, a detailed sequence analysis, and biochemical assays infer its molecular function to be 3-hexulose-6-phosphate isomerase (PHI). In the dissimilatory ribulose monophosphate (RuMP) cycle, ribulose-5-phosphate is coupled to formaldehyde by the 3-hexulose-6-phosphate synthase (HPS), yielding hexulose-6-phosphate, which is then isomerized to fructose-6-phosphate by the enzyme 3-hexulose-6-phosphate isomerase. MJ1247 is an alpha/beta structure consisting of a five-stranded parallel beta sheet flanked on both sides by alpha helices, forming a three-layered alpha-beta-alpha sandwich. The fold represents the nucleotide binding motif of a flavodoxin type. MJ1247 is a tetramer in the crystal and in solution and each monomer has a folding similar to the isomerase domain of glucosamine-6-phosphate synthase (GlmS).     

Structural basis for phosphomannose isomerase activity in phosphoglucose isomerase from Pyrobaculum aerophilum: a subtle difference between distantly related enzymes

The crystal structure of a dual-specificity phosphoglucose/phosphomannose isomerase from the crenarchaeon Pyrobaculum aerophilum (PaPGI/PMI) has been determined in complex with glucose 6-phosphate at 1.16 A resolution and with fructose 6-phosphate at 1.5 A resolution. Subsequent modeling of mannose 6-phosphate (M6P) into the active site of the enzyme shows that the PMI activity of this enzyme may be due to the additional space imparted by a threonine. In PGIs from bacterial and eukaryotic sources, which cannot use M6P as a substrate, the equivalent residue is a glutamine. The increased space may permit rotation of the C2-C3 bond in M6P to facilitate abstraction of a proton from C2 by Glu203 and, after a further C2-C3 rotation of the resulting cis-enediolate, re-donation of a proton to C1 by the same residue. A proline residue (in place of a glycine in PGI) may also promote PMI activity by positioning the C1-O1 region of M6P. Thus, the PMI reaction in PaPGI/PMI probably uses a cis-enediol mechanism of catalysis, and this activity appears to arise from a subtle difference in the architecture of the enzyme, compared to bacterial and eukaryotic PGIs.