Crystallographic studies of bacterial sialyltransferases
Creators: name: Chiu, Cecilia P. C.
Publisher: name: University of British Columbia
Year: 2011

description: Sialic acids terminate oligosaccharide chains on the surfaces of mammalian cells and many microbial species, often playing critical biological roles in recognition and adherence. The enzymes which transfer the sialic acid moiety to these key terminal positions are known as sialyltransferases. Despite their important biological roles very little is understood about the mechanism of action or molecular structure of these enzymes. Campylobacter jejuni, a highly prevalent food-borne pathogen that causes acute gastroenteritis in humans, contains two versions of a sialyltransferase: a monofunctional α-2,3-sialyltransferase Cst-I and a bifunctional α-2,3/8-sialyltransferase Cst-II. Both of these enzymes are responsible for lipooligosaccharides (LOS) sialylation to camouflage the bacterial surface from the host, and thus evade the immune system. In addition, sialylated-glycoconjugates on C. jejuni often mimic human gangliosides, contributing to the molecular basis of Guillain-Barré syndrome, an autoimmune disease of the peripheral nervous system that often develops post-infection. The sialyltransferase reaction is believed to proceed through an inversion mechanism, catalyzing the transfer of sialic acid from CMP-N-acetylneuraminic acid onto different acceptors. This thesis is to understand through high-resolution structural characterization, site specific mutagenesis and kinetic analysis, the mechanism of the glycosyl transfer(s) in both monofunctional and bifunctional Csts. Crystals of Cst-II were obtained and the complex structures with bound CMP, inert donor sugar analogue CMP-3-fluoro-N-acetylneuraminic acid (CMP-3FNeu5Ac) and inhibitor CDP were solved using MAD phasing from incorporated selenomethionines. Work within this study represents the first known structure of a sialyltransferase. Based on the position of the substrates, the active site of Cst-II has been elucidated. Site-directed mutagenesis of conserved residues in the active site was performed and mutants were characterized using enzyme kinetics. A reaction mechanism was proposed based on the kinetic assay. A directed evolution methodology was designed for glycosyltransferases using Cst-II as the model system. A single mutation, F91Y was found to substantially increase the reaction rate of the enzyme with a fluorescent-coupled acceptor, bodipy-lactose. The crystal structure of this Cst-II F91Y mutant was solved and it revealed an unexpected flip of the tyrosine side chain of Y91 from the core of the enzyme into the solvent region, exposing a hydrophobic pocket which seems to be capable of accommodating the bodipy ring structure. Together with kinetic analyses, the crystallographic study was able to explain the observed increase in the catalytic rate for this novel sugar acceptor. A monofunctional variant of Cst, Cst-I, also isolated from Campylobacter jejuni, was characterized crystallographically and kinetically. The conservation of active site residues supports the proposed mechanism for GT-42 sialyltransferases. The complex structure of the Cst-I enzyme with the donor analogue CMP-3FNeu5Ac provides a platform for molecular modeling of various acceptors into the active sites of Cst-I and Cst-II. The modeling shed lights upon the understanding of differences in substrate specificity. The structures of these complexes will be used as templates to design therapeutic inhibitors against this common human pathogen.