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J. Biol. Chem., Vol. 280, Issue 6, 4792-4802, February 11, 2005

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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, and Warren W. Wakarchuk**

From the Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R6 and the Departments of Biochemistry and ^||Microbiology and Immunology, McGill University, Montréal, Québec H3A 1A4, Canada

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 µM, whereas those for UDP-Glc and UDP-Gal are 780 and 784 µM. 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 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.

Received for publication, July 9, 2004 , and in revised form, October 26, 2004.

^* This work was supported by the National Research Council Genomics and Health Initiative. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Present address: Dept. of Microbiology, University of Guelph, Ontario N1G 2W1, Canada.

^** To whom correspondence should be addressed. Tel.: 613-952-4299; E-mail: warren.wakarchuk{at}nrc-cnrc.gc.ca.

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