Functional Characterization of Gne (UDP-
            <i>N</i>
            -Acetylglucosamine- 4-Epimerase), Wzz (Chain Length Determinant), and Wzy (O-Antigen Polymerase) of
            <i>Yersinia enterocolitica</i>
            Serotype O:8

UDP-galactose 4-epimerase (GALE) interconvertsUDP-galactose and UDP-glucose in the final step of the Leloir pathway. Unlike the Escherichia coli enzyme, human GALE (hGALE) also efficiently interconverts a larger pair of substrates: UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. The basis of this differential substrate specificity has remained obscure. Recently, however, x-ray crystallographic data have both predicted essential active site residues and suggested that differential active site cleft volume may be a key factor in determining GALE substrate selectivity. We report here a direct test of this hypothesis. In brief, we have created four substituted alleles: S132A, Y157F, S132A/Y157F, and C307Y-hGALE. While the first three substitutions were predicted to disrupt catalytic activity, the fourth was predicted to reduce active site cleft volume, thereby limiting entry or rotation of the larger but not the smaller substrate. All four alleles were expressed in a null-background strain of Saccharomyces cerevisiae and characterized in terms of activity with regard to both UDP-galactose and UDP-N-acetylgalactosamine. The S132A/Y157F and C307Y-hGALE proteins were also overexpressed in Pichia pastoris and purified for analysis. In all forms tested, the Y157F, S132A, and Y157F/S132A-hGALE proteins each demonstrated a complete loss of activity with respect to both substrates. In contrast, the C307Y-hGALE demonstrated normal activity with respect to UDP-galactose but complete loss of activity with respect to UDP-N-acetylgalactosamine. Together, these results serve to validate the wild-type hGALE crystal structure and fully support the hypothesis that residue 307 acts as a gatekeeper mediating substrate access to the hGALE active site.

Section: Expression Of Wild-type and Substituted Human Gale Enzymes Imentioning

confidence: 92%

Determinants of Function and Substrate Specificity in Human UDP-galactose 4′-Epimerase

Schulz

Watson

Sanders

et al. 2004

“…UDP-GlcNAc/GalNAc 4-epimerase activity but displays weak UDP-Glc/Gal 4-epimerase activities (19,20).…”

Section: Discussionmentioning

confidence: 99%

“…This epimerization of UDPGlcNAc and UDP-GalNAc by the C. jejuni GalE has not been demonstrated before. Because GalE from C. jejuni possesses UDP-N-acetylglucosaminyl 4-epimerase activity, it will henceforth be designated Gne (UDP-GlcNAc 4-epimerase), after the enzymes with the same function in B. subtilis, E. coli, and Y. enterocolitica (20,37,43).…”

Section: Sequence Analysis Of Gale From C Jejuni Strain Nctcmentioning

confidence: 99%

A Single Bifunctional UDP-GlcNAc/Glc 4-Epimerase Supports the Synthesis of Three Cell Surface Glycoconjugates in Campylobacter jejuni

Bernatchez

Szymanski

Ishiyama

et al. 2005

111

The major cell-surface carbohydrates (lipooligosaccharide, capsule, and glycoprotein N-linked heptasaccharide) of Campylobacter jejuni NCTC 11168 contain Gal and/or GalNAc residues. GalE is the sole annotated UDP-glucose 4-epimerase in this bacterium. The presence of GalNAc residues in these carbohydrates suggested that GalE might be a UDP-GlcNAc 4-epimerase. GalE was shown to epimerize UDP-Glc and UDP-GlcNAc in coupled assays with C. jejuni glycosyltransferases and in sugar nucleotide epimerization equilibria studies. Thus, GalE possesses UDP-GlcNAc 4-epimerase activity and was renamed Gne. The K m(app) values of a purified MalE-Gne fusion protein for UDP-GlcNAc and UDP-GalNAc are 1087 and 1070 M, whereas those for UDP-Glc and UDP-Gal are 780 and 784 M. The k cat and k cat /K m(app) values were three to four times higher for UDP-GalNAc and UDP-Gal than for UDP-GlcNAc and UDP-Glc. The comparison of the kinetic parameters of MalE-Gne to those of other characterized bacterial UDPGlcNAc 4-epimerases indicated that Gne is a bifunctional UDP-GlcNAc/Glc 4-epimerase. The UDP sugarbinding site of Gne was modeled by using the structure of the UDP-GlcNAc 4-epimerase WbpP from Pseudomonas aeruginosa. Small differences were noted, and these may explain the bifunctional character of the C. jejuni Gne. In a gne mutant of C. jejuni, the lipooligosaccharide was shown by capillary electrophoresis-mass spectrometry to be truncated by at least five sugars. Furthermore, both the glycoprotein N-linked heptasaccharide and capsule were no longer detectable by high resolution magic angle spinning NMR. These data indicate that Gne is the enzyme providing Gal and GalNAc residues with the synthesis of all three cell-surface carbohydrates in C. jejuni NCTC 11168.

“…GalE, a group 1 epimerase from K. pneumoniae (33), was not capable of using either UDP-D-GlcNAcA or UDP-D-GalNAcA, but this is not surprising because GalE could not utilize N-acetylated UDP-sugars as substrates (19,37). Gne, a group 2 epimerase from Y. enterocolitica (34), was able to epimerize UDP-D-GlcNAcA at a rather low conversion rate, i.e. only 14% conversion to UDP-D-GalNAcA was observed at equilibrium.…”

Section: Substratementioning

confidence: 99%

Flagellin Glycosylation in Pseudomonas aeruginosa PAK Requires the O-antigen Biosynthesis Enzyme WbpO

Miller

Matewish

McNally

et al. 2008

Pseudomonas aeruginosa PAK (serotype O6) produces a single polar, glycosylated flagellum composed of a-type flagellin. To determine whether or not flagellin glycosylation in this serotype requires O-antigen genes, flagellin was isolated from the wild type, three O-antigen-deficient mutants wbpL, wbpO, and wbpP, and a wbpO mutant complemented with a plasmid containing a wild-type copy of wbpO. Flagellin from the wbpO mutant was smaller (42 kDa) than that of the wild type (45 kDa), or other mutants strains, and exhibited an altered isoelectric point (pI 4.8) when compared with PAK flagellin (pI 4.6). These differences were because of the truncation of the glycan moiety in the wbpO-flagellin. Thus, flagellin glycosylation in P. aeruginosa PAK apparently requires a functional WbpO but not WbpP. Because WbpP was previously proposed to catalyze a metabolic step in the biosynthesis of B-band O-antigen that precedes the action of WbpO, these results prompted us to reevaluate the two-step pathway catalyzed by WbpO and WbpP. Results from WbpO-WbpP-coupled enzymatic assays showed that either WbpO or WbpP is capable of initiating the two-step pathway; however, the kinetic parameters favored the WbpO reaction to occur first, converting UDP-N-acetyl-D-glucosamine to UDP-N-acetyl-D-glucuronic acid prior to the conversion to UDP-N-acetyl-D-galacturonic acid by WbpP. This is the first report to show that a C4 epimerase could utilize UDP-N-acetylhexuronic acid as a substrate.