Two tomato GDP-D-mannose epimerase isoforms involved in ascorbate biosynthesis play specific roles in cell wall biosynthesis and development.

GDP-D-mannose epimerase (GME, EC 5.1.3.18) converts GDP-D-mannose to GDP-L-galactose, and is considered to be a central enzyme connecting the major ascorbate biosynthesis pathway to primary cell wall metabolism in higher plants. Our previous work demonstrated that GME is crucial for both ascorbate and cell wall biosynthesis in tomato. The aim of the present study was to investigate the respective role in ascorbate and cell wall biosynthesis of the two SlGME genes present in tomato by targeting each of them through an RNAi-silencing approach. Taken individually SlGME1 and SlGME2 allowed normal ascorbate accumulation in the leaf and fruits, thus suggesting the same function regarding ascorbate. However, SlGME1 and SlGME2 were shown to play distinct roles in cell wall biosynthesis, depending on the tissue considered. The RNAi-SlGME1 plants harbored small and poorly seeded fruits resulting from alterations of pollen development and of pollination process. In contrast, the RNAi-SlGME2 plants exhibited vegetative growth delay while fruits remained unaffected. Analysis of SlGME1- and SlGME2-silenced seeds and seedlings further showed that the dimerization state of pectin rhamnogalacturonan-II (RG-II) was altered only in the RNAi-SlGME2 lines. Taken together with the preferential expression of each SlGME gene in different tomato tissues, these results suggest sub-functionalization of SlGME1 and SlGME2 and their specialization for cell wall biosynthesis in specific tomato tissues.

D-glucuronyl C5-epimerase cell type specifically affects angiogenesis pathway in different prostate cancer cells.

D-glucuronyl C5-epimerase (GLCE) is involved in breast and lung carcinogenesis as a potential tumor suppressor gene, acting through inhibition of tumor angiogenesis and invasion/metastasis pathways. However, in prostate tumors, increased GLCE expression is associated with advanced disease, suggesting versatile effects of GLCE in different cancers. To investigate further the potential cancer-promoting effect of GLCE in prostate cancer, GLCE was ectopically re-expressed in morphologically different LNCaP and PC3 prostate cancer cells. Transcriptional profiles of normal PNT2 prostate cells, LNCaP, PC3 and DU145 prostate cancer cells, and GLCE-expressing LNCaP and PC3 cells were determined. Comparative analysis revealed the genes whose expression was changed in prostate cancer cells compared with normal PNT2 cells, and those differently expressed between the cancer cell lines (ACTA2, IL6, SERPINE1, TAGLN, SEMA3A, and CDH2). GLCE re-expression influenced mainly angiogenesis-involved genes (ANGPT1, SERPINE1, IGF1, PDGFB, TNF, IL8, TEK, IFNA1, and IFNB1) but in a cell type-specific manner (from basic deregulation of angiogenesis in LNCaP cells to significant activation in PC3 cells). Invasion/metastasis pathway was also affected (MMP1, MMP2, MMP9, S100A4, ITGA1, ITGB3, ERBB2, and FAS). The obtained results suggest activation of angiogenesis as a main molecular mechanism of pro-oncogenic effect of GLCE in prostate cancer. GLCE up-regulation plus expression pattern of a panel of six genes, discriminating morphologically different prostate cancer cell sub-types, is suggested as a potential marker of aggressive prostate cancer.

Decreased myo-inositol to chiro-inositol (M/C) ratios and increased M/C epimerase activity in PCOS theca cells demonstrate increased insulin sensitivity compared to controls.

Previous studies from our and other labs have shown that insulin resistance is associated with an inositol imbalance of excess myo-inositol and deficient chiro-inositol together with a deficiency of myo-inositol to chiro-inositol epimerase in vivo and in vitro. In this report, we utilized well characterized theca cells from normal cycling women, with normal insulin sensitivity, and theca cells from women with polycystic ovary syndrome (PCOS), with increased insulin sensitivity to examine the myo-inositol to chiro-inisitol (M/C) ratio and the myo-inositol to chiro-inositol epimerase activity. PCOS theca cells with increased insulin sensitivity were specifically used to investigate whether the inositol imbalance and myo-inositol to chiro-inositol epimerase are regulated in a similar or the opposite direction than that observed in insulin resistant cells. The results of these studies are the first to demonstrate that in insulin sensitive PCOS theca cells the inositol imbalance goes in the opposite direction to that observed in insulin resistant cells, and there is a decreased M/C ratio and an increased myo-inositol to chiro-inositol epimerase activity. Further biochemical and genetic studies will probe the mechanisms involved.

Lack of aldose 1-epimerase in Hypocrea jecorina (anamorph Trichoderma reesei): a key to cellulase gene expression on lactose.

The heterodisaccharide lactose (1,4-O-beta-D-galactopyranosyl-D-glucose) induces cellulase formation in the ascomycete Hypocrea jecorina (= Trichoderma reesei). Lactose assimilation is slow, and the assimilation of its beta-D-galactose moiety depends mainly on the operation of a recently described reductive pathway and depends less on the Leloir pathway, which accepts only alpha-D-galactose. We therefore reasoned whether galactomutarotase [aldose 1-epimerase (AEP)] activity might limit lactose assimilation and thus influence cellulase formation. We identified three putative AEP-encoding genes (aep1, aep2, aep3) in H. jecorina, of which two encoded intracellular protein (AEP1 and AEP2) and one encoded an extracellular protein (AEP3). Although all three were transcribed, only the aep3 transcript was detected on lactose. However, no mutarotase activity was detected in the mycelia, their cell walls, or the extracellular medium during growth on lactose. Therefore, the effect of galactomutarotase activity on lactose assimilation was studied with H. jecorina strains expressing the C-terminal galactose mutarotase part of the Saccharomyces cerevisiae Gal10. These strains showed increased growth on lactose in a gene copy number-dependent manner, although their formation of extracellular beta-galactosidase activity and transcription of the genes encoding the first steps in the Leloir and the reductive pathway was similar to the parental strain QM9414. Cellulase gene transcription on lactose dramatically decreased in these strains, but remained unaffected during growth on cellulose. Our data show that cellulase induction in H. jecorina by lactose requires the beta-anomer of D-galactose and reveal the lack of mutarotase activity during growth on lactose as an important key for cellulase formation on this sugar.

Male-like sexual behavior of female mouse lacking fucose mutarotase.

BACKGROUND: Mutarotases are recently characterized family of enzymes that are involved in the anomeric conversions of monosaccharides. The mammalian fucose mutarotase (FucM) was reported in cultured cells to facilitate fucose utilization and incorporation into protein by glycosylation. However, the role of this enzyme in animal has not been elucidated. RESULTS: We generated a mutant mouse specifically lacking the fucose mutarotase (FucM) gene. The FucM knockout mice displayed an abnormal sexual receptivity with a drastic reduction in lordosis score, although the animals were fertile due to a rare and forced intromission by a typical male. We examined the anteroventral periventricular nucleus (AVPv) of the preoptic region in brain and found that the mutant females showed a reduction in tyrosine hydoxylase positive neurons compared to that of a normal female. Furthermore, the mutant females exhibited a masculine behavior, such as mounting to a normal female partner as well as showing a preference to female urine. We found a reduction of fucosylated serum alpha-fetoprotein (AFP) in a mutant embryo relative to that of a wild-type embryo. CONCLUSIONS: The observation that FucM-/- female mouse exhibits a phenotypic similarity to a wild-type male in terms of its sexual behavior appears to be due to the neurodevelopmental changes in preoptic area of mutant brain resembling a wild-type male. Since the previous studies indicate that AFP plays a role in titrating estradiol that are required to consolidate sexual preference of female mice, we speculate that the reduced level of AFP in FucM-/- mouse, presumably resulting from the reduced fucosylation, is responsible for the male-like sexual behavior observed in the FucM knock-out mouse.

The Azotobacter vinelandii AlgE mannuronan C-5-epimerase family is essential for the in vivo control of alginate monomer composition and for functional cyst formation.

The industrially widely used polysaccharide alginate is a co-polymer of beta-D-mannuronic acid and alpha-L-guluronic acid (G), and the G residues originate from a polymer-level epimerization process catalysed by mannuronan C-5-epimerases. In the genome of the alginate-producing bacterium Azotobacter vinelandii genes encoding one periplasmic (AlgG) and seven secreted such epimerases (AlgE1-7) have been identified. Here we report the generation of a strain (MS163171) in which all the algE genes were inactivated by deletion (algE1-4 and algE6-7) or interruption (algE5). Shake flask-grown MS163171 produced a polymer containing less than 2% G (algG still active), while wild-type alginates contained 25% G. Interestingly, addition of proteases to the MS163171 growth medium resulted in a strong increase in the chain lengths of the alginates produced. MS163171 was found to be unable to form functional cysts, which is a desiccation-resistant differentiated form developed by A. vinelandii under certain environmental conditions. We also generated mutants carrying interruptions in each separate algE gene, and a strain containing algE5 only. Studies of these mutants indicated that single algE gene inactivations, with the exception of algE3, did not affect the fractional G content much. However, for all strains tested the alginate composition varied somewhat as a response to the growth conditions.

The interaction of phosphorylase a with D-glucose displays alpha-stereospecificity.

Half-maximal inhibition of phosphorylase a required a much lower concentration of alpha-D-glucose (4 mM) than of the beta-anomer (14 mM) and of 1-deoxyglucose (about 25 mM). beta-D-Glucose was almost ineffective at concentrations of 1-2.5 mM, but at 50 mM the two anomers were equipotent. A similar picture emerged when the stimulatory effects of the glucose anomers and of 1-deoxyglucose were investigated on the inactivation of phosphorylase by phosphorylase phosphatase. However, upon addition of either glucose anomer (5-20 mM) to a suspension of isolated hepatocytes, the inactivation of phosphorylase occurred at the same rate. It is shown that, in the latter conditions, the rate of intracellular mutarotation considerably exceeds the rate of glucose transport. This results presumably in a rapid anomeric equilibrium in the liver cells.

D-glucuronyl C5-epimerase acts in dorso-ventral axis formation in zebrafish.

BACKGROUND: Heparan sulfate (HS) is an ubiquitous component of the extracellular matrix that binds and modulates the activity of growth factors, cytokines and proteases. Animals with defective HS biosynthesis display major developmental abnormalities however the processes that are affected remain to be defined. D-glucuronyl-C5-epimerase (Glce) is a key HS chain modifying enzyme that catalyses the conversion of glucuronic acid into iduronic acid, a biosynthetic step that enhances HS biological activity. In this study the role of Glce during early zebrafish development has been investigated. RESULTS: Two Glce-like proteins (Glce-A and -B) are expressed in zebrafish at all times. They are the products of two distinct genes that, based on chromosomal mapping, are both orthologues of the same single human gene. Transcripts for both proteins were detected in fertilized zebrafish embryos prior to the onset of zygotic transcription indicating their maternal origin. At later developmental stages the epimerases are expressed widely throughout gastrulation and then become restricted to the hindbrain at 24 h post-fertilization. By monitoring the expression of well characterized marker genes during gastrulation, we have found that misexpression of Glce causes a dose-dependent expansion of the ventral structures, whereas protein knockdown using targeted antisense morpholino oligonucleotides promotes axis dorsalization. The ventralizing activity of Bmp2b is enhanced by Glce overexpression whereas Glce knockdown impairs Bmp2b activity. CONCLUSION: Glce activity is an important determinant of of dorso-ventral axis formation and patterning in zebrafish. In particular Glce acts during gastrulation by affecting Bmp-mediated cell specification. The results obtained further corroborate the concept that HS encodes information that affect morphogenesis during early vertebrate development.

Fructoselysine 3-epimerase, an enzyme involved in the metabolism of the unusual Amadori compound psicoselysine in Escherichia coli.

The frl (fructoselysine) operon encodes fructoselysine 6-kinase and fructoselysine 6-phosphate deglycase, allowing the conversion of fructoselysine into glucose 6-phosphate and lysine. We now show that a third enzyme encoded by this operon catalyses the metal-dependent reversible interconversion of fructoselysine with its C-3 epimer, psicoselysine. The enzyme can be easily assayed through the formation of tritiated water from [3-3H]fructoselysine. Psicoselysine supports the growth of Escherichia coli, causing the induction of the three enzymes of the frl operon. No growth on fructoselysine or psicoselysine was observed with Tn5 mutants in which the putative transporter (FrlA) or fructoselysine 6-phosphate deglycase (FrlB) had been inactivated, indicating the importance of the frl operon for the metabolism of both substrates. The ability of E. coli to grow on psicoselysine suggests the occurrence of this unusual Amadori compound in Nature.

Hexuronyl C5-epimerases in alginate and glycosaminoglycan biosynthesis.

The sugar residues in most polysaccharides are incorporated as their corresponding monomers during polymerization. Here we summarize the three known exceptions to this rule, involving the biosynthesis of alginate, and the glycosaminoglycans, heparin/heparan sulfate and dermatan sulfate. Alginate is synthesized by brown seaweeds and certain bacteria, while glycosaminoglycans are produced by most animal species. In all cases one of the incorporated sugar monomers are being C5-epimerized at the polymer level, from D-mannuronic acid to L-guluronic acid in alginate, and from D-glucuronic acid to L-iduronic acid in glycosaminoglycans. Alginate epimerization modulates the mechanical properties of seaweed tissues, whereas in bacteria it seems to serve a wide range of purposes. The conformational flexibility of iduronic acid units in glycosaminoglycans promotes apposition to, and thus functional interactions with a variety of proteins at cell surfaces and in the extracellular matrix. In the bacterium Azotobacter vinelandii the alginates are being epimerized at the cell surface or in the extracellular environment by a family of evolutionary strongly related modular type and Ca(2+)-dependent epimerases (AlgE1-7). Each of these enzymes introduces a specific distribution pattern of guluronic acid residues along the polymer chains, explaining the wide structural variability observed in alginates isolated from nature. Glycosaminoglycans are synthesized in the Golgi system, through a series of reactions that include the C5-epimerization reaction along with extensive sulfation of the polymers. The single, Ca(2+)-independent, epimerase in heparin/heparan sulfate biosynthesis and the Ca(2+)-dependent dermatan sulfate epimerase(s) also generate variable epimerization patterns, depending on other polymer-modification reactions. The alginate and heparin epimerases appear unrelated at the amino acid sequence level, and have probably evolved through independent evolutionary pathways; however, hydrophobic cluster analysis indicates limited similarity. Seaweed alginates are widely used in industry, while heparin is well established in the clinic as an anticoagulant.

Glucuronyl C5-epimerase an enzyme converting glucuronic acid to iduronic acid in heparan sulfate/heparin biosynthesis.

The glucuronyl C5 epimerase (HSepi) is one of the modification enzymes involved in biosynthesis of heparan sulfate (HS) and heparin, catalyzing the epimerization of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA) at polymer level. IdoA is critical for HS and heparin to interact with protein ligands, because of its flexible conformation. Although the enzyme recognizes both GlcA and IdoA as substrates catalyzing a reversible reaction of the hexuronic acids in vitro, the reaction appears irreversible in vivo. Targeted interruption of the gene, Glce, in mice resulted in neonatal lethality accompanied with kidney agenesis, premature lung, and skeletal malformations, demonstrating that the single gene coded enzyme is essential for animal development. Elimination of the enzyme resulted in abnormal HS and heparin structure that completely lacks IdoA residues. Loss of 2-O-sulfation due to lacking IdoA in HS chains appears compensated by increased N- and O-sulfation of the glucosamine residues. Recombinant HSepi is used to generate HS/heparin related compounds having potential to be used for therapeutic purposes.

Characterization and role of fucose mutarotase in mammalian cells.

L-Fucose for mammalian glycosylation contains an anomeric carbon atom generating alpha- and beta-L-fucoses. Based on sequence comparison of mouse and human homologs with the prokaryotic fucose mutarotases (FucU) characterized previously, we investigated their function in mammalian cells. By nuclear magnetic resonance (NMR) measurement with saturation difference analysis, the purified mammalian mutarotases were demonstrated to be involved in an interconversion between the two anomeric forms with comparable efficiency as that of the Escherichia coli FucU. The mouse gene was widely expressed in various tissues and cell lines, including kidney, liver, and pancreas, although expression was marginal in muscle and testis. By generating stably expressed cell lines for mutarotase genes in HepG2, it was shown that fucose incorporations into cellular proteins were increased as demonstrated by an incorporation of radiolabeled fucose into the cells. Furthermore, intracellular levels of GDP-L-fucose, measured with high performance liquid chromatography (HPLC), were enhanced by an overproduction of cellular mutarotase, which was reversed by gene silencing of mutarotase based on RNA interference. The results suggest that the mammalian mutarotase is functional in facilitated incorporation of fucose through the salvage pathway.

Role of Gne and GalE in the virulence of Aeromonas hydrophila serotype O34.

The mesophilic Aeromonas hydrophila AH-3 (serotype O34) strain shows two different UDP-hexose epimerases in its genome: GalE (EC 3.1.5.2) and Gne (EC 3.1.5.7). Similar homologues were detected in the different mesophilic Aeromonas strains tested. GalE shows only UDP-galactose 4-epimerase activity, while Gne is able to perform a dual activity (mainly UDP-N-acetyl galactosamine 4-epimerase and also UDP-galactose 4-epimerase). We studied the activities in vitro of both epimerases and also in vivo through the lipopolysaccharide (LPS) structure of A. hydrophila gne mutants, A. hydrophila galE mutants, A. hydrophila galE-gne double mutants, and independently complemented mutants with both genes. Furthermore, the enzymatic activity in vivo, which renders different LPS structures on the mentioned A. hydrophila mutant strains or the complemented mutants, allowed us to confirm a clear relationship between the virulence of these strains and the presence/absence of the O34 antigen LPS.

The UDP N-acetylgalactosamine 4-epimerase gene is essential for mesophilic Aeromonas hydrophila serotype O34 virulence.

Mesophilic Aeromonas hydrophila strains of serotype O34 typically express smooth lipopolysaccharide (LPS) on their surface. A single mutation in the gene that codes for UDP N-acetylgalactosamine 4-epimerase (gne) confers the O(-) phenotype (LPS without O-antigen molecules) on a strain in serotypes O18 and O34, but not in serotypes O1 and O2. The gne gene is present in all the mesophilic Aeromonas strains tested. No changes were observed for the LPS core in a gne mutant from A. hydrophila strain AH-3 (serotype O34). O34 antigen LPS contains N-acetylgalactosamine, while no such sugar residue forms part of the LPS core from A. hydrophila AH-3. Some of the pathogenic features of A. hydrophila AH-3 gne mutants are drastically reduced (serum resistance or adhesion to Hep-2 cells), and the gne mutants are less virulent for fish and mice compared to the wild-type strain. Strain AH-3, like other mesophilic Aeromonas strains, possess two kinds of flagella, and the absence of O34 antigen molecules by gne mutation in this strain reduced motility without any effect on the biogenesis of both polar and lateral flagella. The reintroduction of the single wild-type gne gene in the corresponding mutants completely restored the wild-type phenotype (presence of smooth LPS) independently of the O wild-type serotype, restored the virulence of the wild-type strain, and restored motility (either swimming or swarming).

Identification of the Vibrio vulnificus wbpP gene and evaluation of its role in virulence.

A wbpP gene encoding a putative UDP-N-acetyl-D-glucosamine C(4) epimerase was identified and cloned from Vibrio vulnificus. The functions of the wbpP gene, assessed by the construction of an isogenic mutant and by evaluating its phenotype changes, demonstrated that WbpP is essential in both the pathogenesis and the capsular polysaccharide biosynthesis of V. vulnificus.

Roles for UDP-GlcNAc 2-epimerase/ManNAc 6-kinase outside of sialic acid biosynthesis: modulation of sialyltransferase and BiP expression, GM3 and GD3 biosynthesis, proliferation, and apoptosis, and ERK1/2 phosphorylation.

Roles for UDP-GlcNAc 2-epimerase/ManNAc 6-kinase (GNE) beyond controlling flux into the sialic acid biosynthetic pathway by converting UDP-GlcNAc to N-acetylmannosamine are described in this report. Overexpression of recombinant GNE in human embryonic kidney (HEK AD293) cells led to an increase in mRNA levels for ST3Gal5 (GM3 synthase) and ST8Sia1 (GD3 synthase) as well as the biosynthetic products of these sialyltransferases, the GM3 and GD3 gangliosides. Conversely, down-regulation of GNE by RNA interference methods had the opposite, but consistent, effect of lowering ST3Gal5 and ST8Sia1 mRNAs and reducing GM3 and GD3 levels. Control experiments ensured that GNE-mediated changes in sialyltransferase expression and ganglioside biosynthesis were not the result of altered flux through the sialic acid pathway. Interestingly, exogenous GM3 and GD3 also changed the expression of GNE and led to reduced ST3Gal5 and ST8Sia1 mRNA levels, demonstrating a reciprocating feedback mechanism where gangliosides regulate upstream biosynthetic enzymes. Cellular responses to the GNE-mediated changes in ST3Gal5 and ST8Sia1 expression and GM3 and GD3 levels were investigated next. Conditions that led to reduced ganglioside production (e.g. short hairpin RNA exposure) stimulated proliferation, whereas conditions that resulted in increased ganglioside levels (e.g. recombinant GNE and exogenous gangliosides) led to reduced proliferation with a concomitant increase in apoptosis. Finally, changes to BiP expression and ERK1/2 phosphorylation consistent with apoptosis and proliferation, respectively, were observed. These results provide examples of specific biochemical pathways, other than sialic acid metabolism, that are influenced by GNE.

Heparan sulfate C5-epimerase is essential for heparin biosynthesis in mast cells.

Biosynthesis of heparin, a mast cell-derived glycosaminoglycan with widespread importance in medicine, has not been fully elucidated. In biosynthesis of heparan sulfate (HS), a structurally related polysaccharide, HS glucuronyl C5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) to L-iduronic acid (IdoA) residues. We have generated Hsepi-null mouse mutant mast cells, and we show that the same enzyme catalyzes the generation of IdoA in heparin and that 'heparin' lacking IdoA shows a distorted O-sulfation pattern.

Analysis of Campylobacter jejuni capsular loci reveals multiple mechanisms for the generation of structural diversity and the ability to form complex heptoses.

We recently demonstrated that Campylobacter jejuni produces a capsular polysaccharide (CPS) that is the major antigenic component of the classical Penner serotyping system distinguishing Campylobacter into >60 groups. Although the wide variety of C. jejuni serotypes are suggestive of structural differences in CPS, the genetic mechanisms of such differences are unknown. In this study we sequenced biosynthetic cps regions, ranging in size from 15 to 34 kb, from selected C. jejuni strains of HS:1, HS:19, HS:23, HS:36, HS:23/36 and HS:41 serotypes. Comparison of the determined cps sequences of the HS:1, HS:19 and HS:41 strains with the sequenced strain, NCTC11168 (HS:2), provides evidence for multiple mechanisms of structural variation including exchange of capsular genes and entire clusters by horizontal transfer, gene duplication, deletion, fusion and contingency gene variation. In contrast, the HS:23, HS:36 and HS:23/36 cps sequences were highly conserved. We report the first detailed structural analysis of 81-176 (HS:23/36) and G1 (HS:1) and refine the previous structural interpretations of the HS:19, HS:23, HS:36 and HS:41 serostrains. For the first time, we demonstrate the commonality and function of a second heptose biosynthetic pathway for Campylobacter CPS independent of the pathway for lipooligosaccharide (LOS) biosynthesis and identify a novel heptosyltransferase utilized by this alternate pathway. Furthermore, we show the retention of two functional heptose isomerases in Campylobacter and the sharing of a phosphatase for both LOS and CPS heptose biosynthesis.

The NeuC protein of Escherichia coli K1 is a UDP N-acetylglucosamine 2-epimerase.

The K1 capsule is an essential virulence determinant of Escherichia coli strains that cause meningitis in neonates. Biosynthesis and transport of the capsule, an alpha-2,8-linked polymer of sialic acid, are encoded by the 17-kb kps gene cluster. We deleted neuC, a K1 gene implicated in sialic acid synthesis, from the chromosome of EV36, a K-12-K1 hybrid, by allelic exchange. Exogenously added sialic acid restored capsule expression to the deletion strain (DeltaneuC), confirming that NeuC is necessary for sialic acid synthesis. The deduced amino acid sequence of NeuC showed similarities to those of UDP-N-acetylglucosamine (GlcNAc) 2-epimerases from both prokaryotes and eukaryotes. The NeuC homologue from serotype III Streptococcus agalactiae complements DeltaneuC. We cloned the neuC gene into an intein expression vector to facilitate purification. We demonstrated by paper chromatography that the purified neuC gene product catalyzed the formation of [2-(14)C]acetamidoglucal and [N-(14)C]acetylmannosamine (ManNAc) from UDP-[(14)C]GlcNAc. The formation of reaction intermediate 2-acetamidoglucal with the concomitant release of UDP was confirmed by proton and phosphorus nuclear magnetic resonance spectroscopy. NeuC could not use GlcNAc as a substrate. These data suggest that neuC encodes an epimerase that catalyzes the formation of ManNAc from UDP-GlcNAc via a 2-acetamidoglucal intermediate. The unexpected release of the glucal intermediate and the extremely low rate of ManNAc formation likely were a result of the in vitro assay conditions, in which a key regulatory molecule or protein was absent.