Investigating diversity in O antigen polysaccharide glycosyltransferases: structures, mechanisms, and regulation
O antigen polysaccharides (OPSs) are repetitive sugar polymers that decorate the cell surface of Gram-negative bacteria. OPSs facilitate interactions with potential hosts (both pathogenically and symbiotically), protect against bacteriophage predators and form a barrier to harmful small molecules. These interactions are dictated by the specific composition and length of the OPS, which show extreme variation between and even within species. A large class of enzymes, known as glycosyltransferases (GTs), is responsible for polymerizing the OPS from individual monosaccharides, linked together in a regio- and stereo-specific manner, often to discrete lengths. The research in this thesis explores several aspects of O antigen biosynthesis, with a focus on diverse examples of GTs which produce them. First, using the β-Kdo GT WbbBGT99 from Raoultella terrigena, I thoroughly detail the first unambiguous use of a double displacement mechanism in GTs through a combination of structural and biochemical approaches. Second, a novel type llb system for OPS chain-length regulation is proposed for Rhizobium tropici CIAT 899, based on biochemical analysis of the functional enzymes present in the cluster. This strategy substitutes a β-helix spacer domain for α-helical coiled-coils in key enzyme, ORF3. Finally, the fucosyltransferase, ORF6, from the R. tropici OPS cluster was the subject of a thorough structure-function analysis which revealed a new metal-independent GT family. Overall, this thesis describes the catalytic mechanisms for two divergent GTs in detail, advancing our understanding of GT mechanisms, and highlights the initial characterization of the R. tropici OPS biosynthesis cluster, providing evidence for a novel strategy of glycan length control.