Bacterial biofilms are often untreatable in the human body. Biofilm formation commonly depends on exopolysaccharides that serve to facilitate bacterial interactions within the biofilm and increase antimicrobial resistance. In Escherichia coli, the PgaCD enzyme is critically involved in exopolysaccharide synthesis. PgaCD forms an inner membrane associated glycosyltransferase complex responsible for polymerization of UDP-GlcNAc to form the poly-β-(1,6)-N-acetylglucosamine (PNAG) polysaccharide. Inhibitors that specifically block the catalytic activity of PgaCD have the potential to prevent or substantially inhibit biofilm formation. The combination of multiple kinetic isotope effect measurements with quantum chemical density functional theory (DFT) calculations is an efficient and comprehensive method capable of probing changes in bonding in enzyme transition state structures. The experimental data collected can serve to guide the design of potent enzyme transition state analogue inhibitors, which mimic the charge and geometry of the transition state structure of enzyme catalyzed reactions and bind to their target enzymes with affinities orders of magnitude higher than the native enzyme substrates. Here we present a one-pot chemoenzymatic method to prepare isotopically labeled UDP-GlcNAc from commercially available labeled GlcNAc. This method has been used to prepare [1′′-13C] and [2′′-13C]-UDP-GlcNAc in milligram quantities. These substrates are being used to measure multiple kinetic isotope effects for the glycosyltransferase reaction catalyzed by PgaCD. Identifying the transition state and the subsequent synthesis of a transition state analogue will enable the study in vivo of the role of exopolysaccharide synthesis during the formation of bacterial biofilms.

Kinetic isotope effects and transition state analysis for Escherichia coli poly-N-acetylglucosamine synthase

2019 Spring National Meeting

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