This project aims to expand the mechanistic understanding of the initial membrane-associated steps of bacterial glycoconjugate biosynthesis. Membrane protein structures are critically underrepresented in the protein data bank (PDB), and remain difficult targets for purification, characterization and mechanistic analysis. The products of these biosynthetic pathways are required for both bacterial viability and virulence and are attractive targets for antimicrobial design. Examples include capsular polysaccharide (CPS), cell wall teichoic acid, lipopolysaccharide (LPS) and N- and O-linked glycoproteins. The first membrane-bound step of glycoconjugate biosynthesis is catalyzed by phosphoglycosyl transferases (PGTs). This class of enzymes transfers a sugar from an NDP-sugar onto an undecaprenyl phosphate lipid anchor. Different PGTs exhibit different substrate selectivity, the molecular determinants of which remain unknown. The first structure of a PGT, PglC from Campylobacter concisus was solved recently, but crystallized in a conformation in which the active site is open and unliganded. Both Styrene maleic acid copolymer (SMALP) and traditional detergents will be used to solubilize and purify PGTs of differing substrate selectivity. Chemoenzymatic synthesis will be used to generate UDP-sugar substrates for PGTs such as UDP-diNAcBac and UDP-fucose. A rapid, luminescence-based assay will be used to characterize solubilized targets and synthesized substrates. Lipid cubic phase (LCP) methods will facilitate crystallization of SMALP solubilized targets that have never left a lipid bilayer. Synthetic substrates will be utilized for soaking or cocrystallization experiments. These experiments will broaden our understanding of PGT structure-function relationships. Data will also inform efforts to develop inhibitors, as PGTs remain an underexplored area for the development of antimicrobial and antivirulence agents.
In the age of antibiotic resistance, novel targets for antimicrobial development are sorely needed. Bacterial viability and virulence are dependent on a variety of glycoconjugates found appended to the cell surface. A conserved step in the biosynthesis of many of these glycoconjugates is the transfer of a sugar phosphate from a UDP-sugar to a lipid carrier, catalyzed by phosphoglycosyl transferases (PGTs). Here, emerging techniques in membrane protein purification and crystallization alongside rapid PGT assay technologies will be used to biochemically and structurally characterize several PGTs from human pathogens. This project aims to further the understanding of the structure-function relationships of PGTs, which will inform future efforts to target these enzymes with antimicrobials.
