| Small molecules that affect specific protein functions can be valuable tools for dissecting complex cellular processes. Here, we report the use of small molecules to probe the mechanism of DNA gyrase and the regulation of peptidoglycan biosynthesis.; Bacterial topoisomerase, or DNA gyrase, is an essential enzyme that is involved in transcription and DNA replication. We synthesized and evaluated dimeric derivatives of the coumarin antibiotic novobiocin hoping that they would bind to the dimeric enzyme DNA gyrase with improved affinity and specificity. In the course of these studies, we developed a novel one-step purification of DNA gyrase.; Vancomycin, an inhibitor of peptidoglycan synthesis, functions by binding to bacterial cell wall precursors. Resistance occurs when these precursors change. Derivatives of vancomycin containing a lipid substituent attached to the sugar moiety 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 trans glycosylation step of peptidoglycan biosynthesis. By attaching transglycosylase inhibitors to the vancomycin aglycone, we were able to synthesize derivatives of vancomycin with improved activity against vancomycin resistant bacteria. To gain more information about how glycolipid derivatives of vancomycin kill bacteria, we obtained and analyzed mutant Escherichia coli that were resistant to these derivatives, but not to vancomycin itself. We were able to identify the gene that was responsible for this phenotype. This provides a genetic basis for the activity differences between vancomycin and the glycolipid derivatives. The E. coli mutants were not only resistant to glycolipid derivatives of vancomycin, but also failed to die in the presence of these compounds. We conclude that the mutated gene is responsible for the rapid death of E. coli cells upon treatment with glycolipid derivatives of vancomycin. |
