Lipid II, transglycosylases, and clinically relevant bacterial strains: Tools for understanding glycopeptide antibiotic mode of action

Vancomycin is one of the few widely available antibiotics used to treat methicillin-resistant Gram-positive infections. It is a glycopeptide antibiotic that inhibits peptidoglycan biosynthesis by binding to bacterial cell wall precursors and resistance occurs when these precursors change. Lipid-linked vancomycin derivatives can overcome resistance by accessing a second mechanism that is independent of precursor binding. The Kahne lab has suggested that this mechanism, which is not available to vancomycin, involves inhibition of the transglycosylation step of peptidoglycan biosynthesis. Understanding vancomycin and its derivatives mode of action requires the development of a transglycosylase assay and the efficient synthesis of substrate Lipid II.; There are many difficulties in studying these enzymes. Transglycosylases have a transmembrane domain which makes purification difficult, and the substrate is detergent-like which makes its synthesis and handling a challenge. In order to study these enzymes an improved synthetic route to Lipid II was developed to provide access to milligram quantities of substrate. This synthesis is convergent and allows us to make substrate analogs of differing lipid and peptide configurations. New methodology was developed for forming pyrophosphate bonds using stannous chloride which improves the yield and decreases side product formation. With a convergent synthesis in place we generated a series of substrates with which to study transglycosylase enzymes.; E. coli membrane preparations were used to establish a transglycosylase assay and we determined that the best substrate contained a C35 lipid chain. An E. coli PBP1b assay was then developed and kinetic parameters of an apparent Km for Lipid II of 2 muM and a kcat of 0.07 s,-1 were obtained. Kinetic studies were conducted with a series of glycopeptides and glycopeptide derivatives. We found that lipid-linked vancomycin derivatives with a damaged precursor binding pocket nevertheless inhibit E. coli PBP1b, demonstrating that these compounds interact directly with the enzyme. These findings support the hypothesis that lipid-linked vancomycin derivatives overcome resistance by targeting transglycosylases. Therefore, using multiple sequence alignment tools homologs in S. aureus and E. facaelis were identified, and these enzymes were overexpressed and found to be active. Experiments to determine the ability of glycopeptide derivatives to inhibit these transglycosylases are ongoing.