A Multitarget Assay for Inhibitors of Membrane-Associated Steps of Peptidoglycan Biosynthesis

✍ Scribed by Maria D.F.S. Barbosa; Harold O. Ross; Milton C. Hillman; Raymond P. Meade; Michael G. Kurilla; David L. Pompliano

• Publisher: Elsevier Science
• Year: 2002
• Tongue: English
• Weight: 150 KB
• Volume: 306
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2001.5691

✦ Synopsis

Peptidoglycan synthesis begins in the cytoplasm with the condensation of UDP-N-acetyl glucosamine (UDP-GlcNAc) and phosphoenolpyruvate catalyzed by UDP-N-acetylglucosamine enolpyruvoyl transferase. UDP-GlcNAc is also utilized as substrate for the glycosyltransferase MurG, a membrane-bound enzyme that catalyzes the production of lipid II. Membranes from Escherichia coli cells overproducing MurG support peptidoglycan formation at a rate approximately fivefold faster than membranes containing wild-type levels of MurG. Conditions have been optimized for the production of large amounts of membranes with increased levels of MurG, allowing the development of an assay suitable for high-throughput screening of large compound libraries. The quality of the purified membranes was assessed by electron microscopy and also by testing cross-linked peptidoglycan production. Moreover, kinetic studies allowed the determination of optimal concentrations of the substrates and membranes to be utilized for maximum sensitivity of the assay. Using a 96-well assay format, the IC50 values for vancomycin, tunicamycin, flavomycin, and bacitracin were 1.1 M, 0.01 g/ml, 0.03 g/ml, and 0.7 g/ml, respectively.