Scientific Publications by FDA Staff

Glycobiology 2018 Feb 1;28(2):100-7

Interaction of Neisseria meningitidis Group X N-Acetylglucosamine-1-phosphotransferase with Its Donor Substrate.

Ming SA, Cottman-Thomas E, Black NC, Chen Y, Veeramachineni V, Peterson DC, Chen X, Tedaldi LM, Wagner GK, Cai C, Linhardt RJ, Vann WF

Neisseria meningitidis Group X is an emerging cause of bacterial meningitis in the Sub-Saharan Africa. The capsular polysaccharide of Group X is a homopolymer of N-acetylglucosamine a(1-4) phosphate and is a vaccine target for prevention of disease associated with this meningococcal serogroup. We have demonstrated previously that the formation of the polymer is catalyzed by a phosphotransferase which transfers N- acetylglucosamine-1-phosphate from UDP-N-acetylglucosamine to the 4- hydroxyl of the N-acetylglucosamine on the non-reducing end of the growing chain. In this study, we use substrate analogs of UDP-GlcNAc to define the enzyme/donor substrate interactions critical for catalysis. Our kinetic analysis of the phosphotransferase reaction is consistent with a sequential mechanism of substrate addition and product release. We used novel uracil modified analogs designed by Wagner et al. enabled us to assess whether the CsxA-catalyzed reaction is consistent with a donor dependent conformational change. As expected with this model for glycosyltransferases, UDP-GlcNAc analogs with bulky uracil modifications are not substrates but are inhibitors. An analog with a smaller iodo uracil substitution is a substrate and a less potent inhibitor. Moreover, our survey of analogs with modifications on the N- acetylglucosamine residue of the sugar nucleotide donor highlights the importance of substituents at C-2 and C-4 of the sugar residue. The hydroxyl group at C4 and the structure of the acyl group at C2 are very important for specificity and substrate interactions during the polymerization reaction. While most analogs modified at C-2 were inhibitors, acetamido analogs were also substrates suggesting the importance of the carbonyl group.