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

Stéphane Bernatchez,Christine M Szymanski,Noboru Ishiyama,Jianjun Li,Harold C Jarrell,Peter C Lau,Albert M Berghuis,N Martin Young,Warren W Wakarchuk

doi:10.1074/jbc.m407767200

Stéphane Bernatchez, Christine M Szymanski + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m407767200

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Abstract

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 Km(app) values of a purified MalE-Gne fusion protein for UDP-GlcNAc and UDP-GalNAc are 1087 and 1070 microm, whereas those for UDP-Glc and UDP-Gal are 780 and 784 microm. The kcat and kcat/Km(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 UDP-GlcNAc 4-epimerases indicated that Gne is a bifunctional UDP-GlcNAc/Glc 4-epimerase. The UDP sugar-binding 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.

Highlights

The major cell-surface carbohydrates of Campylobacter jejuni NCTC 11168 contain Gal and/or GalNAc residues
Sequence Analysis of GalE from C. jejuni Strain NCTC 11168 —The Cj1131c gene was amplified by PCR from purified C. jejuni NCTC 11168 genomic DNA and cloned in the expression vector pCWoriϩ (28) to generate the plasmid pCPG6 (Table II)
The C. jejuni NCTC 11168 GalE shares 98% identity with GalE from C. jejuni 81116

Summary

EXPERIMENTAL PROCEDURES

The reactions were started with the addition of an aliquot of CJL30 (positive and negative controls) or CJL30 and PL2 ϩ pCPG6 lysates (coupled assays). The Gne ϩ MalE-CgtB assays were performed in 50 mM MES, pH 6.0, 10 mM MnCl2, 1 mM dithiothreitol, 1 mM UDP-Gal (or UDP-Glc when Gne was present), and 0.5 mM GM2-FCHASE.. The reactions were started with the addition of an aliquot of PL2 ϩ pCJL20 (positive and negative controls) or PL2 ϩ pCJL20 and PL2 ϩ pCPG6 lysates (coupled assays). Epimerization of UDP Sugars by the C. jejuni Gne and Capillary Electrophoresis Quantification—Reactions were performed at 37 °C in a total volume of 30 ␮l in 50 mM Tris-HCl, pH 8.0, with 1 mM of UDP sugar (UDP-GlcNAc, UDP-GalNAc, UDP-Glc, or UDP-Gal) and 15 ␮l of cell lysate of PL2 ϩ pCPG6. The sample spinning rate was 3000 Ϯ 10 Hz, and all other acquisition and data processing parameters were as described previously (5)

RESULTS

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