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J. Biol. Chem., Vol. 280, Issue 21, 20902-20908, May 27, 2005

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Biochemical Characterization of the Campylobacter jejuni Cj1294, a Novel UDP-4-keto-6-deoxy-GlcNAc Aminotransferase That Generates UDP-4-amino-4,6-dideoxy-GalNAc^*

Ravinder Kaur Obhi and Carole Creuzenet, Recipient of a University Faculty Award from the Natural Sciences and Engineering Research Council of Canada, and of a Premier's Research Excellence Award (Ontario)

From the Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1, Canada

Campylobacter jejuni produces multiple glycoproteins whose glycans contain 4-amino 6-deoxy sugars or their derivatives, such as diacetamidobacillosamine or pseudaminic acid. Because the proteoglycans contribute to bacterial virulence and their constitutive sugars are not commonly found in humans, inhibitors developed against the enzymes that are responsible for their biosynthesis could be novel therapeutic targets to fight this important food-borne pathogen. The biosynthesis of diacetamidobacillosamine is anticipated to involve a sugar nucleotide C6 dehydratase, a C4 aminotransferase and an acetyltransferase. We have identified a set of genes (cj1293, cj1294, and cj1298) potentially encoding one of each enzymatic activity, and demonstrated earlier that Cj1293 was a UDP-GlcNAc-specific C6 dehydratase. Others have shown that Cj1293 was involved in protein glycosylation. Here, we report on our investigation of the potential activity of Cj1294 as a sugar nucleotide C4 aminotransferase. Our biochemical characterization of overexpressed and purified protein shows that Cj1294 is a pyridoxal phosphate-dependent aminotransferase specific for UDP-4-keto-6-deoxy-GlcNAc that uses preferentially glutamic acid as an amino donor. A detailed physicokinetic study of Cj1294 was performed to determine the K_m of 1.28 ± 0.2 mM and k_cat of 11.5 ± 1.3 min^-1. Also, two residues essential for protein stability and activity, Arg²²⁸ and Lys¹⁸¹, respectively, were identified by site-directed mutagenesis. Finally, we demonstrated by NMR analysis of purified reaction product that Cj1294 produces UDP-4-amino-4,6-dideoxy-GalNAc. These results indicate that Cj1294 is involved in the biosynthesis of diacetamidofucosamine, a C4 epimer of diacetamidobacillosamine not yet described in C. jejuni proteoglycans, suggesting that the composition of C. jejuni proteoglycans is more variable than anticipated.

Received for publication, December 8, 2004 , and in revised form, March 24, 2005.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY573603.

^* This work was supported by operating Grant MOP-62775 from the Canadian Institutes of Health Research (to C. C.). 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.

To whom correspondence should be addressed: University of Western Ontario, Dept. of Microbiology and Immunology, DSB 3031, N6A 5C1, London, ON, Canada. Tel.: 519-661-3204; Fax: 519-661-3499; E-mail: ccreuzen{at}uwo.ca.

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