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.
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, Arg(228) and Lys(181), 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.
Obhi
Ravinder Kaur
RK
Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1, Canada.
Creuzenet
Carole
C
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D001120:Arginine; D001426:Bacterial Proteins; D016123:Campylobacter jejuni / Q000201:enzymology*; D003001:Cloning, Molecular; D019281:Dimerization; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008239:Lysine; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D000637:Transaminases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014536:Uridine Diphosphate N-Acetylgalactosamine / Q000031:analogs & derivatives* / Q000378:metabolism*
C502751:2,4-diacetamido-2,4,6-trideoxyglucopyranose; D001426:Bacterial Proteins; D011994:Recombinant Proteins; C502749:UDP-4-amino-4,6-dideoxy-N-acetylgalactosamine; D011732:Pyridoxal Phosphate; D014536:Uridine Diphosphate N-Acetylgalactosamine; D001120:Arginine; C502750:Cj1294 protein, Campylobacter jejuni; D000637:Transaminases; D008239:Lysine; D000117:Acetylglucosamine