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

Abstract

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.

Deletion of a previously uncharacterized flagellar-hook-length control gene fliK modulates the σ 54-dependent regulon in Campylobacter jejuni

“…Genes in the Cj1293-Cj1298 cluster have recently been strongly implicated in synthesis of sugars involved in flagellin glycosylation (Obhi & Creuzenet, 2005), and detailed analysis in strain 81176 has further defined the functions of many of the remaining genes (Guerry et al, 2006). Thus, a strong case can be made for co-regulation of flagellin-modification genes with the rod and hook genes described above, implying that the flagellin glycosylation mechanism may be assembled at some point after the export machinery is installed, and possibly at the same time as rod and hook construction.…”

Cj1121c, a Novel UDP-4-keto-6-deoxy-GlcNAc C-4 Aminotransferase Essential for Protein Glycosylation and Virulence in Campylobacter jejuni

“…The harvested cells were kept at Ϫ20°C until needed. The enzyme purification was performed by metal chelation as described before (27). The eluted protein was incubated with PLP before dialysis in 100 mM Hepes, pH 7.5.…”

“…Otherwise, it was kept frozen at Ϫ20°C in the presence of 20% glycerol. Expression and purification of Cj1293 and Cj1294 were performed as described previously (24,27). SDS-PAGE analysis and Western blotting with anti-histidine antibody (Amersham Biosciences) were done according to standard procedures.…”

“…The cj1293 gene is part of an operon that also encodes an aminotransferase (Cj1294) and a putative N-acetyltransferase (Cj1298). We have demonstrated that Cj1294 is a C-4 aminotransferase that uses the reaction product generated by Cj1293 as a substrate, which was the very first aminotransferase with such substrate specificity to be characterized (27). Not suspecting that a change of chirality occurred during dehydration, we had determined previously by NMR that Cj1294 generated UDP-4-amino-4,6-dideoxy-GalNAc (27), which is not a DAB precursor.…”

“…We have demonstrated that Cj1294 is a C-4 aminotransferase that uses the reaction product generated by Cj1293 as a substrate, which was the very first aminotransferase with such substrate specificity to be characterized (27). Not suspecting that a change of chirality occurred during dehydration, we had determined previously by NMR that Cj1294 generated UDP-4-amino-4,6-dideoxy-GalNAc (27), which is not a DAB precursor. By taking the change of chirality at C-5 into account, the NMR assignments had to be revisited so that the reaction product of Cj1294 is UDP-4-amino-4,6-dideoxy-␤-LAltNAc (Fig.…”