Campylobacter jejuni PglH is a single active site processive polymerase that utilizes product inhibition to limit sequential glycosyl transfer reactions.
Asparagine-linked protein glycosylation is essential for the virulence of the human gut mucosal pathogen Campylobacter jejuni . The heptasaccharide that is transferred to proteins is biosynthesized via the glycosyltransferase-catalyzed addition of sugar units to an undecaprenyl diphosphate-linked carrier. Genetic studies on the heptasaccharide assembly enzymes have shown that PglH, which transfers three terminal N-acetyl-galactosamine (GalNAc) residues to the carrier polyisoprene, is essential for chick colonization by C. jejuni . While it is now clear that PglH catalyzes multiple transfer reactions, the mechanism whereby the reactions cease after the addition of just three GalNAc residues has yet to be understood. To address this issue, a series of mechanistic biochemical studies was conducted with purified native PglH. This enzyme was found to follow a processive mechanism under initial rate conditions; however, product inhibition and product accumulation led to PglH release of intermediate products prior to complete conversion to the native ultimate product. Point mutations of an essential EX(7)E sequence motif were used to demonstrate that a single active site was responsible for all three transferase reactions, and a homology model with the mannosyltransferase PimA, from Mycobacteria smegmatis , establishes the requirement of the EX(7)E motif in catalysis. Finally, increased binding affinity with increasing glycan size is proposed to provide PglH with a counting mechanism that does not allow the transfer of more than three GalNAc residues. These results provide important mechanistic insights into the function of the glycosyl transfer polymerase that is related to the virulence of C. jejuni .
Troutman
Jerry M
JM
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Imperiali
Barbara
B
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D016123:Campylobacter jejuni / Q000201:enzymology* / Q000378:metabolism; D020134:Catalytic Domain; D016695:Glycosyltransferases / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D011135:Polysaccharides, Bacterial / Q000378:metabolism*
D001426:Bacterial Proteins; D011135:Polysaccharides, Bacterial; D016695:Glycosyltransferases