Elimination of 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid 9-phosphate synthase activity from human N-acetylneuraminic acid 9-phosphate synthase by a single mutation

The most commonly occurring sialic acid Neu5Ac (N-acetylneuraminic acid) and its deaminated form, KDN (2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid), participate in many biological functions. The human Neu5Ac-9-P (Neu5Ac 9-phosphate) synthase has the unique ability to catalyse the synthesis of not only Neu5Ac-9-P but also KDN-9-P (KDN 9-phosphate). Both reactions are catalysed by the mechanism of aldol condensation of PEP (phosphoenolpyruvate) with sugar substrates, ManNAc-6-P (N-acetylmannosamine 6-phosphate) or Man-6-P (mannose 6-phosphate). Mouse and putative rat Neu5Ac-9-P synthases, however, do not show KDN-9-P synthase activity, despite sharing high sequence identity (>95%) with the human enzyme. Here, we demonstrate that a single mutation, M42T, in human Neu5Ac-9-P synthase can abolish the KDN-9-P synthase activity completely without compromising the Neu5Ac-9-P synthase activity. Saturation mutagenesis of Met42 of the human Neu5Ac-9-P synthase showed that the substitution with all amino acids except leucine retains only the Neu5Ac-9-P synthase activity at levels comparable with the wild-type enzyme. The M42L mutant, like the wild-type enzyme, showed the additional KDN-9-P synthase activity. In the homology model of human Neu5Ac-9-P synthase, Met42 is located 22 A (1 A=0.1 nm) away from the substrate-binding site and the impact of this distant residue on the enzyme functions is discussed.     

Molecular cloning and expression of the mouse N-acetylneuraminic acid 9-phosphate synthase which does not have deaminoneuraminic acid (KDN) 9-phosphate synthase activity

A cDNA of the mouse homologue of Escherichia coli N-acetylneuraminic acid (Neu5Ac) synthase (neuB gene product) was cloned by the PCR-based method. The mouse homologue consists of 359 amino acids, and the cDNA sequence displays 33% identity to that of the E. coli Neu5Ac synthase. The recombinant mouse homologue which is transiently expressed in HeLa cells does not exhibit the Neu5Ac synthase activity, which catalyzes condensation of phosphoenolpyruvate (PEP) and N-acetylmannosamine (ManNAc) to synthesize Neu5Ac, but the Neu5Ac 9-phosphate (Neu5Ac-9-P) synthase activity, which catalyzes condensation of PEP and ManNAc 6-phosphate (ManNAc-6-P) to synthesize Neu5Ac-9-P. Thus, the mouse homologue of E. coli Neu5Ac synthase is the Neu5Ac-9-P synthase. The Neu5Ac-9-P synthase is a cytosolic enzyme and ubiquitously distributed in mouse various tissues. Notably, the Neu5Ac-9-P synthase can not catalyze the synthesis of deaminoneuraminic acid (KDN) or KDN-9-P from PEP and Man or ManNAc-6-P, thus suggesting that the enzyme is not involved in the synthesis of KDN. This is consistent with the previous observation that only a very low activity to synthesize KDN is found in mouse B16 cells [Angata, T., et al. (1999) Biochem. Biophys. Res. Commun. 261, 326-331].     

Cloning, expression, and characterization of sialic acid synthases

The most commonly occurring sialic acid, N-acetylneuraminic acid, is the repeating unit in polysialic acid chain of human neuronal cell adhesion molecule as well as in capsular polysialic acid of neuroinvasive bacteria, Escherichia coli K1 and Neisseria meningitidis. Sialic acid synthesis and polymerization occur in slightly different pathways in animals and bacteria. N-Acetylneuraminic acid (NeuNAc) is synthesized by the condensation of phosphoenolpyruvate and N-acetylmannosamine by NeuNAc synthase in bacteria. The mammalian homologue N-acetylneuraminic acid-9-phosphate (NeuNAc-9-P) synthase uses N-acetylmannosamine-6-phosphate in the condensation reaction to produce NeuNAc-9-P. Both subfamilies of sialic acid synthases possess N-terminal triosephosphate isomerase barrel domain and C-terminal antifreeze protein domain. We report cloning of the genes, expression, purification, and characterization of human NeuNAc-9-P synthase and N. meningitidis NeuNAc synthase. Stability of the purified enzymes and effects of pH and temperature on their activities were evaluated. Enzyme kinetics and preliminary mutagenesis experiments reveal the importance of C-terminal antifreeze protein domain and a conserved cysteine residue for the enzyme activities.     

Solution structure of the antifreeze-like domain of human sialic acid synthase

The structure of the C-terminal antifreeze-like (AFL) domain of human sialic acid synthase was determined by NMR spectroscopy. The structure comprises one alpha- and two single-turn 3(10)-helices and two beta-strands, and is similar to those of the type III antifreeze proteins. Evolutionary trace analyses of the type III antifreeze protein family suggested that the class-specific residues in the human and bacterial AFL domains are important for their substrate binding, while the class-specific residues of the fish antifreeze proteins are gathered on the ice-binding surface.     

Biosynthesis of KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid). Identification and characterization of a KDN-9-phosphate synthetase activity from trout testis.

Although the deaminoneuraminic acid or KDN glycotope (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) is expressed in glycoconjugates that range in evolutionary diversity from bacteria to man, there is little information as to how this novel sugar is synthesized. Accordingly, biosynthetic studies were initiated in trout testis, an organ rich in KDN, to determine how this sialic acid is formed. These studies have shown that the pathway consists of the following three sequential reactions: 1) Man + ATP --> Man-6-P + ADP; 2) Man-6-P + PEP --> KDN-9-P + P(i); 3) KDN-9-P --> KDN + P(i). Reaction 1, catalyzed by a hexokinase, is the 6-O-phosphorylation of mannose to form D-mannose 6-phosphate (Man-6-P). Reaction 2, catalyzed by KDN-9-phosphate (KDN-9-P) synthetase, condenses Man-6-P and phosphoenolpyruvate (PEP) to form KDN-9-P. Reaction 3, catalyzed by a phosphatase, is the dephosphorylation of KDN-9-P to yield free KDN. It is not known if a kinase specific for Man (Reaction 1) and a phosphatase specific for KDN-9-P (Reaction 3) may exist in tissues actively synthesizing KDN. In this study, the KDN-9-P synthetase, an enzyme that has not been previously described, was identified as at least one key enzyme that is specific for the KDN biosynthetic pathway. This enzyme was purified 50-fold from rainbow trout testis and characterized. The molecular weight of the enzyme was estimated to be about 80,000, and activity was maximum at neutral pH in the presence of Mn(2+). N-Acetylneuraminic acid 9-phosphate (Neu5Ac-9-P) synthetase, which catalyzes the condensation of N-acetyl-D-mannosamine 6-phosphate and phosphoenol-pyruvate to produce Neu5Ac-9-P, was co-purified with the KDN-9-P synthetase. Substrate competition experiments revealed, however, that syntheses of KDN-9-P and Neu5Ac-9-P were catalyzed by two separate synthetase activities. The significance of these studies takes on added importance with the recent discovery that the level of free KDN is elevated in human fetal cord but not matched adult red blood cells and in ovarian cancer cells (Inoue, S., Lin, S-L., Chang, T., Wu, S-H., Yao, C-W., Chu, T-Y., Troy, F. A., II, and Inoue, Y. (1998) J. Biol. Chem. 273, 27199-27204). This unexpected finding emphasizes the need to understand more fully the role that free KDN and KDN-glycoconjugates may play in normal hematopoiesis and malignancy.     

Purification and characterization of N-acetylneuraminic acid-9-phosphate synthase from rat liver

Sialic acids are a group of carboxylated amino sugars important for a variety of cellular functions. N-Acetylneuraminic acid (Neu5Ac) is the predominant sialic acid in nature. Neu5Ac-9-phosphate synthase catalyzes the formation of Neu5Ac-9-phosphate from N-acetylmannosamine-6-phosphate and phosphoenolpyruvate. Neu5Ac-9-phosphate synthase was purified 11,700-fold from rat liver cytosol to apparent homogeneity by ammonium sulfate precipitation, chromatography on hydroxylapatite, phenyl-Sepharose, MonoQ, and finally gel filtration. SDS-PAGE and gel filtration chromatography indicated that the enzyme is a dimer composed of 37-kDa subunits. Analysis of trypic peptides by MALDI-TOF MS verified a high sequence similarity to the corresponding murine enzyme. The K(m) values of Neu5Ac-9-phosphate synthase were 35 microM for N-acetylmannosamine-6-phosphate and 100 microM for phosphoenolpyruvate. The enzyme displayed an absolute requirement for divalent cations, Mn(2+), Fe(2+), and Mg(2+) being the most effective. In contrast to human Neu5Ac-9-phosphate synthase, the rat enzyme did not utilize mannose-6-phosphate in the synthesis of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid 9-phosphate. Neu5Ac-9-phosphate synthase was inactivated by the sulfhydryl modifying reagents, 5,5'-dithio-bis (2-nitrobenzoic acid) and N-ethylmaleimide, and protected from inactivation by the presence of the substrate phosphoenolpyruvate, but not by the presence of N-acetylmannosamine-6-phosphate, showing that at least one cysteine residue is located in the active site of the enzyme.     

Comparison of backbone dynamics of the type III antifreeze protein and antifreeze-like domain of human sialic acid synthase

Antifreeze proteins (AFPs) are found in a variety of cold-adapted (psychrophilic) organisms to promote survival at subzero temperatures by binding to ice crystals and decreasing the freezing temperature of body fluids. The type III AFPs are small globular proteins that consist of one α-helix, three 3₁₀-helices, and two β-strands. Sialic acids play important roles in a variety of biological functions, such as development, recognition, and cell adhesion and are synthesized by conserved enzymatic pathways that include sialic acid synthase (SAS). SAS consists of an N-terminal catalytic domain and a C-terminal antifreeze-like (AFL) domain, which is similar to the type III AFPs. Despite having very similar structures, AFL and the type III AFPs exhibit very different temperature-dependent stability and activity. In this study, we have performed backbone dynamics analyses of a type III AFP (HPLC12 isoform) and the AFL domain of human SAS (hAFL) at various temperatures. We also characterized the structural/dynamic properties of the ice-binding surfaces by analyzing the temperature gradient of the amide proton chemical shift and its correlation with chemical shift deviation from random coil. The dynamic properties of the two proteins were very different from each other. While HPLC12 was mostly rigid with a few residues exhibiting slow motions, hAFL showed fast internal motions at low temperature. Our results provide insight into the molecular basis of thermostability and structural flexibility in homologous psychrophilic HPLC12 and mesophilic hAFL proteins.     

The crystal and solution studies of glucosamine-6-phosphate synthase from Candida albicans

Glucosamine 6-phosphate (GlcN-6-P) synthase is an ubiquitous enzyme that catalyses the first committed step in the reaction pathway that leads to formation of uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), a precursor of macromolecules that contain amino sugars. Despite sequence similarities, the enzyme in eukaryotes is tetrameric, whereas in prokaryotes it is a dimer. The activity of eukaryotic GlcN-6-P synthase (known as Gfa1p) is regulated by feedback inhibition by UDP-GlcNAc, the end product of the reaction pathway, whereas in prokaryotes the GlcN-6-P synthase (known as GlmS) is not regulated at the post-translational level. In bacteria and fungi the enzyme is essential for cell wall synthesis. In human the enzyme is a mediator of insulin resistance. For these reasons, Gfa1p is a target in anti-fungal chemotherapy and in therapeutics for type-2 diabetes. The crystal structure of the Gfa1p isomerase domain from Candida albicans has been analysed in complex with the allosteric inhibitor UDP-GlcNAc and in the presence of glucose 6-phosphate, fructose 6-phosphate and an analogue of the reaction intermediate, 2-amino-2-deoxy-d-mannitol 6-phosphate (ADMP). A solution structure of the native Gfa1p has been deduced using small-angle X-ray scattering (SAXS). The tetrameric Gfa1p can be described as a dimer of dimers, with each half similar to the related enzyme from Escherichia coli. The core of the protein consists of the isomerase domains. UDP-GlcNAc binds, together with a metal cation, in a well-defined pocket on the surface of the isomerase domain. The residues responsible for tetramerisation and for binding UDP-GlcNAc are conserved only among eukaryotic sequences. Comparison with the previously studied GlmS from E. coli reveals differences as well as similarities in the isomerase active site. This study of Gfa1p focuses on the features that distinguish it from the prokaryotic homologue in terms of quaternary structure, control of the enzymatic activity and details of the isomerase active site.     

Isomerase activity of the C-terminal fructose-6-phosphate binding domain of glucosamine-6-phosphate synthase from Escherichia coli

The isomerase activity of the C-terminal fructose-6P binding domain (residues 241-608) of glucosamine-6-phosphate synthase from Escherichia coli has been studied. The equilibrium constant of the C-terminal domain k(eq) ([glucose-6P]/[fructose-6-P]) = 5.0. A non-competitive product inhibition of the isomerase activity by the reaction product glucose-6-P has been detected. The existence of more than one binding and reaction sites for the substrate fructose-6P on the molecule of glucosamine-6-phosphate synthase can be expected. The fructose-6P binding domain possibly includes a regulatory site, different from the catalytic center of the enzyme.     

Phosphate inhibition of spinach leaf sucrose phosphate synthase as affected by glucose-6-phosphate and phosphoglucoisomerase

The inhibition patterns of inorganic phosphate (Pi) on sucrose phosphate synthase activity in the presence and absence of the allosteric activator glucose-6-P was studied, as well as the effects of phosphoglucoisomerase on fructose-6-P saturation kinetics with and without Pi. In the presence of 5 millimolar glucose-6-P, Pi was a partial competitive inhibitor with respect to both substrates, fructose-6-P and uridine diphosphate glucose. In the absence of glucose-6-P, the inhibition patterns were more complex, apparently because of the interaction of Pi at the activation site as well as the catalytic site. In addition, substrate activation by uridine diphosphate glucose was observed in the absence of effectors. The results suggested that Pi antagonizes glucose-6-P activation of sucrose phosphate synthase by competing with the activator for binding to the modifier site.

The fructose-6-P saturation kinetics were hyperbolic in the absence of phosphoglucoisomerase activity, but became sigmoidal by the addition of excess phosphoglucoisomerase. The sigmoidicity persisted in the presence of Pi, but sucrose phosphate synthase activity was decreased. The apparent sigmoidal response may represent the physiological response of sucrose phosphate synthase to a change in hexose-P concentration because sucrose phosphate synthase operates in the cytosol in the presence of high activities of phosphoglucoisomerase. Thus, the enzymic production of an activator from a substrate represents a unique mechanism for generating sigmoidal enzyme kinetics.

     

Helicobacter pylori 3-deoxy-D-manno-octulosonate-8-phosphate (KDO-8-P) synthase is a zinc-metalloenzyme

3-Deoxy-D-manno-2-octulosonate-8-phosphate (KDO-8-P) synthase catalyzes the aldol-type condensation of phosphoenolpyruvate and D-arabinose-5-phosphate (A-5-P) to produce KDO-8-P and inorganic phosphate. All KDO-8-P synthases, as exemplified by the enzyme from Escherichia coli, were believed not to require a metal cofactor for catalytic activity. However, recent studies have demonstrated that the KDO-8-P synthase from Aquifex aeolicus is a metalloenzyme. Moreover, sequence alignments and phylogenetic analysis of KDO-8-P synthase protein sequences strongly suggested that there is a whole subfamily of KDO-8-P synthases that are also metalloenzymes. One of these putative metalloenzymes is the ortholog from the human pathogen Helicobacter pylori. In order to test this model, we have cloned the kdsa gene encoding H. pylori KDO-8-P synthase, and overexpressed and purified the protein. This enzyme was found to bind one mol Zn/mol monomer, and the removal of this metal by treatment with 2,6-pyridine dicarboxylic acid abolished enzymatic activity. The Zn(2+) in the enzyme could be quantitatively replaced by Cd(2+), which increased the observed k(cat) by approximately 2-fold, and decreased the apparent K(m)(A-5-P) by approximately 6.5-fold. Furthermore, removal of the Zn(2+) from the enzyme did not greatly perturb its circular dichroism spectra. Thus, the divalent metal most likely serves as cofactor directly involved in catalysis.     

Synthesis of 2,3-bisphosphoglycerate synthase in erythroid cells

Antiserum prepared from a rabbit which was immunized with human erythrocyte glycerate-2,3-P2 synthase was found to react with glycerate-2,3-P2 synthase in rabbit erythroid cells. By using this antiserum, it was proved that the specific activity of this enzyme was unchanged during the development of the rabbit erythroid cells. This leads us to conclude that the increased activity of glycerate-2,3-P2 synthase in developing erythroid cells (Narita, H., Ikura, K., Yanagawa, S., Sasaki, R., Chiba, H., Saimyoji, H., and Kumagai, N. (1980) J. Biol. Chem. 255, 5230-5235) is due to the accumulation of enzyme protein. There is at least a 16-fold increase in the level of this protein during development from bone marrow erythroid cells to erythrocytes. The synthesis of glycerate-2,3-P2 synthase was shown to occur in rabbit reticulocytes and bone marrow erythroid cells. These cells were incubated for protein synthesis and the protein synthesized was precipitated with the anti-glycerate-2,3-P2 synthase antiserum and separated on sodium dodecyl sulfate-polyacrylamide gels. The immunoprecipitated product was shown to produce fragments of the same molecular weight after digestion with V8 protease as did the pure glycerate-2,3-P2 synthase. The proportion of glycerate-2,3-P2 synthase synthesis in reticulocytes (0.04% of total protein synthesis) was comparable to the level of this protein in the cells (0.07% of the total protein).     

Long range molecular dynamics study of regulation of eukaryotic glucosamine-6-phosphate synthase activity by UDP-GlcNAc

Glucosamine-6-phosphate (GlcN-6-P) synthase catalyses the first and practically irreversible step in hexosamine metabolism. The final product of this pathway, uridine 5’ diphospho N-acetyl-D-glucosamine (UDP-GlcNAc), is an essential substrate for assembly of bacterial and fungal cell walls. Moreover, the enzyme is involved in phenomenon of hexosamine induced insulin resistance in type II diabetes, which makes it a potential target for antifungal, antibacterial and antidiabetic therapy. The crystal structure of the isomerase domain of GlcN-6-P synthase from human pathogenic fungus Candida albicans, in complex with UDP-GlcNAc has been solved recently but it has not revealed the molecular mechanism of inhibition taking place under UDP-GlcNAc influence, the unique feature of the eukaryotic enzyme. UDP-GlcNAc is a physiological inhibitor of GlcN-6-P synthase, binding about 1 nm away from the active site of the enzyme. In the present work, comparative molecular dynamics simulations of the free and UDP-GlcNAc-bounded structures of GlcN-6-P synthase have been performed. The aim was to complete static X-ray structural data and detect possible changes in the dynamics of the two structures. Results of the simulation studies demonstrated higher mobility of the free structure when compared to the liganded one. Several amino acid residues were identified, flexibility of which is strongly affected upon UDP-GlcNAc binding. Importantly, the most fixed residues are those related to the inhibitor binding process and to the catalytic reaction. The obtained results constitute an important step toward understanding of mechanism of GlcN-6-P synthase inhibition by UDP-GlcNAc molecule.     

Effect of vanadate on the glucose-1,6-bisphosphate synthase and glucose-1,6-bisphosphatase activities of phosphoglucomutase

Phosphoglucomutase, in addition to catalyzing the interconversion of glucose 1-P and glucose 6-P, catalyzes both the synthesis of glucose 1,6-P2 from glucose monophosphate and either fructose 1,6-P2 or glycerate 1,3-P2, and the hydrolysis of glucose 1,6-P2. Vanadate inhibits the mutase activity, activates the synthase activities, and does not affect the phosphatase activity. These effects suggest that the "exchange" step postulated for the phosphoglucomutase pathway is specifically inhibited by vanadate.     

Synthesis and evaluation of C-9 modified N-acetylneuraminic acid derivatives as substrates for N-acetylneuraminic acid aldolase

Several C-9 modified N-acetylneuraminic acid derivatives have been synthesised and evaluated as substrates of N-acetylneuraminic acid aldolase. Simple C-9 acyl or ether modified derivatives of N-acetylneuraminic acid were found to be accepted as substrates by the enzyme, albeit being transformed more slowly than Neu5Ac itself. 1H NMR spectroscopy was used to evaluate the extent of the enzyme catalysed transformation of these compounds. Interestingly, the chain-extended Neu5Ac derivative 16 is not a substrate for N-acetylneuraminate lyase and behaves as an inhibitor of the enzyme.     

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.     

Subcellular localization and tissue distribution of sialic acid-forming enzymes. N-acetylneuraminate-9-phosphate synthase and N-acetylneuraminate 9-phosphatase.

The activities of N-acetylneuraminate 9-phosphate synthase and N-acetylneuraminate 9-phosphatase, the two enzymes involved in the final steps of the biosynthetic pathway of N-acetylneuraminic acid, were measured with the substrates N-acetyl[14C]mannosamine 6-phosphate and N-acetyl[14C]neuraminic acid 9-phosphate respectively. Subcellular localization studies in rat liver indicated that both enzymes are localized in the cytosolic fraction after homogenization in sucrose medium. To test the possibility of misinterpretation due to the hydrolysis of N-acetylneuraminic acid 9-phosphate by non-specific phosphatases, the hydrolysis of various phosphate esters by the cytosolic fraction was tested. Only p-nitrophenyl phosphate was hydrolysed; however, competition studies with N-acetylneuraminic acid 9-phosphate and p-nitrophenyl phosphate indicated that two different enzymes were involved and that no competition existed between the two substrates. In various other rat tissues N-acetylneuraminate-9-phosphate synthase and N-acetylneuraminate 9-phosphatase activities were detected, suggesting that N-acetylmannosamine 6-phosphate is a general precursor for N-acetylneuraminic acid biosynthesis in all the tissues studied.     

Histidine 268 in 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase plays the same role as histidine 202 in 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase

The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAH 7-P) synthase (Phe) is inactivated by diethyl pyrocarbonate (DEPC). The inactivation is first order with respect to enzyme and DEPC concentrations with a pseudo-second order rate constant of inactivation by DEPC of 4.9 +/- 0.8 m(-1) s(-1) at pH 6.8 and 4 degrees C. The dependence of inactivation on pH and the spectral features of enzyme modified at specific pH values imply that both histidine and cysteine residues are modified, which is confirmed by site-directed mutagenesis. Analysis of the chemical modification data indicates that one histidine is essential for activity. DAH 7-P synthase (Phe) is protected against DEPC inactivation by phosphoenolpyruvate, whereas d-erythrose 4-phosphate offers only minimal protection. The conserved residues H-172, H-207, H-268, and H-304 were individually mutated to glycine. The H304G and H207G mutants retain some level of activity, whereas the H268G and H172G mutants are virtually inactive. A comparison of the circular dichroism spectra of wild-type enzyme and the various mutants demonstrates that H-172 may play a structural role. Comparison of the UV spectra of the H268G and wild-type enzymes saturated with Cu(2+) indicates that the metal-binding site of the H268G mutant resembles that of the wild-type enzyme. The residue H-268 may play a catalytic role based on the site-directed mutagenesis and spectroscopic studies. Cysteine 61 appears to influence the pK(a) of H-268 in the wild-type enzyme. The pK(a) of H-268 increases from 6.0 to 7.0 following mutation of C-61 to glycine.     

Shikimate-3-phosphate binds to the isolated N-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the transfer of the enolpyruvyl moiety from phosphoenolpyruvate (PEP) to shikimate-3-phosphate (S3P). Mutagenesis and X-ray crystallography data suggest that the active site of the enzyme is in the cleft between its two globular domains; however, they have not defined which residues are responsible for substrate binding and catalysis. Here we attempt to establish the binding of the substrate S3P to the isolated N-terminal domain of EPSP synthase using a combination of NMR spectroscopy and isothermal titration calorimetry. Our experimental results indicate that there is a saturable and stable conformational change in the isolated N-terminal domain upon S3P binding and that the chemical environment of the S3P phosphorus when bound to the isolated domain is very similar to that of S3P bound to EPSP synthase. We also conclude that most of the free energy of S3P binding to EPSP synthase is contributed by the N-terminal domain.     

Cloning and expression of the human N-acetylneuraminic acid phosphate synthase gene with 2-keto-3-deoxy-D-glycero- D-galacto-nononic acid biosynthetic ability

Sialic acids participate in many important biological recognition events, yet eukaryotic sialic acid biosynthetic genes are not well characterized. In this study, we have identified a novel human gene based on homology to the Escherichia coli sialic acid synthase gene (neuB). The human gene is ubiquitously expressed and encodes a 40-kDa enzyme. The gene partially restores sialic acid synthase activity in a neuB-negative mutant of E. coli and results in N-acetylneuraminic acid (Neu5Ac) and 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) production in insect cells upon recombinant baculovirus infection. In vitro the human enzyme uses N-acetylmannosamine 6-phosphate and mannose 6-phosphate as substrates to generate phosphorylated forms of Neu5Ac and KDN, respectively, but exhibits much higher activity toward the Neu5Ac phosphate product.