StructUral and Biochemical Investigation of PgIF from Campylobacter jejuni Reveals a New Mechanism for a Member of the Short Chain Dehydrogenase/Reductase Superfamily

Within recent years it has become apparent that protein glycosylation is not limited to eukaryotes. Indeed, in Campylobacter jejuni, a Gram-negative bacterium, more than 60 of its proteins are known to be glycosylated. One of the sugars found in such glycosylated proteins is 2,4diacetamido-2,4,6-trideoxy-alpha-D-glucopyranose, hereafter referred to as QuiNAc4-NAc. The pathway for its biosynthesis, initiating with UDP-G1cNAc, requires three enzymes referred to as Pg1F, PglE, and P1gD. The focus of this investigation is on Pg1F, an NAD(+)-dependent sugar 4,6-dehydratase known to belong to the short chain dehydrogenase/reductase (SDR) superfamily. Specifically, PgIF catalyzes the first step in the pathway, namely, the dehydration of UDP-G1cNAc to UDP-2-acetamido-2,6-dideoxy-alpha-D-xylo-hexos-4-ulose. Most members of the SDR super family contain a characteristic signature sequence of YXXXK where the conserved tyrosine functions as a catalytic acid or a base. Strikingly, in Pg1F, this residue is a methionine. Here we describe a detailed structural and functional investigation of PgIF from C. jejuni. For this investigation five X-ray structures were determined to resolutions of 2.0 angstrom or better. In addition, kinetic analyses of the wild-type and site-directed variants were performed. On the basis of the data reported herein, a new catalytic mechanism for a SDR superfamily member is proposed that does not require the typically conserved tyrosine residue.