| Author | Search for: Bernatchez, Stéphane1; Search for: Szymanski, Christine M.1; Search for: Ishiyama, Noboru; Search for: Li, Jianjun1; Search for: Jarrell, Harold C.1; Search for: Lau, Peter C.1; Search for: Berghuis, Albert M.; Search for: Young, N. Martin1; Search for: Wakarchuk, Warren W.1 |
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| Affiliation | - National Research Council Canada. NRC Institute for Biological Sciences
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| Format | Text, Article |
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| Subject | angle; assay; bacteria; bacterial; binding; binding site; Campylobacter jejuni; Canada; capsule; carbohydrates; cells; cell surface; difference; enzymes; equilibrium; glycoconjugates; glycoprotein; glycosyltransferases; high-resolution; kinetic; lipooligosaccharides; magic angle spinning; mutant; N-linked; NMR spectroscopy; parameters; protein; Pseudomonas aeruginosa; residues; resolution; site; structure; sugars; surface; synthesis |
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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 may be an 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) of a purified MalE-Gne fusion protein for UDP-GlcNAc and UDP-GalNAc are 1087M and 1070 M while those for UDP-Glc and UDP-Gal are 780 M and 784 M. The kcat and kcat/ Km(app) 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 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 LOS was shown by CE-MS 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 for the synthesis of all three cell-surface carbohydrates in C. jejuni NCTC 11168 |
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| Publication date | 2005-10-27 |
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| Publisher | American Society for Biochemistry and Molecular Biology |
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| In | |
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| Language | English |
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| Peer reviewed | Yes |
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| NRC number | BERNATCHEZ2005 |
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| NPARC number | 9387849 |
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| Export citation | Export as RIS |
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| Report a correction | Report a correction (opens in a new tab) |
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| Record identifier | ae8a0e04-9758-498a-aa40-a5297614db13 |
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| Record created | 2009-07-10 |
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| Record modified | 2020-04-07 |
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