Structural characterization of essential enzymes involving sialic acid metabolism in bacterial pathogens

Sialic acid plays vital roles in various biological processes including cellular recognition and cell adhesion. The biosynthesis and post-synthetic processing of sialic acid is particularly important to host-pathogen interactions because many virulent bacteria decorate their cell surfaces with sialic acid-containing molecules in order to evade the host’s immune response. Neisseria meningitidis, a highly invasive human pathogen that causes bacterial meningitis, produces a capsular polysaccharide comprised of polysialic acids that protect the bacteria from the host’s immune system by mimicking the sialic acid-containing cell surface structures. The biosynthesis of sialic acid is catalyzed by sialic acid synthase NeuB. We report the structural and biochemical analysis of the first potent inhibitor of sialic acid synthase from N. meningitidis. The inhibitor was synthesized as a mixture of stereoisomers, which mimics the tetrahedral intermediate of the NeuB reaction. Based on the crystallographic and kinetic analysis of the inhibitor binding, an improved mechanism is proposed. Capsular polysaccharides of certain strains of N. meningitidis can be further acetylated by sialic acid-specific O-acetyltransferases, a modification that correlates with the virulence in bacterial infection. In the second part of the thesis, we report the first kinetic and structural analysis of bacterial sialic acid O-acetyltransferase OatWY from N. meningitidis. Crystals of OatWY were obtained in complex with either CoA, acetyl-CoA, or nonhydrolyzable donor analogue S-(2-oxopropyl)-CoA. Structural analysis in combination with kinetic and mutagenesis studies elucidates the mechanistic features and substrate specificity of this enzyme. Campylobacter jejuni, a leading causative agent of bacterial diarrhea and gastroenteritis, expresses sialylated lipooligosaccharide, which mimics the carbohydrate structure of human gangliosides. Sialyltransferase Cst-II is the enzyme responsible for the lipooligosaccharide sialylation in C. jejuni as a means of evading the host's immune system. The last part of the thesis describes the first ternary complex of Cst-II with the donor analogue CMP and the terminal trisaccharide (Neu5Ac-α-2,3-Gal-β-1,3-GalNAc) of its natural acceptor. Site-directed mutagenesis of acceptor binding residues was performed and mutants were characterized by enzyme kinetics. Our results reveal the structural basis for the binding of a physiologically relevant natural acceptor and provide additional insight into the mechanism and acceptor specificity of this enzyme.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat