PMID:12069409
| Citation |
Barbosa, MD, Ross, HO, Hillman, MC, Meade, RP, Kurilla, MG and Pompliano, DL (2002) A multitarget assay for inhibitors of membrane-associated steps of peptidoglycan biosynthesis. Anal. Biochem. 306:17-22 |
|---|---|
| Abstract |
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 microM, 0.01 microg/ml, 0.03 microg/ml, and 0.7 microg/ml, respectively. |
| Links |
PubMed Online version:10.1006/abio.2001.5691 |
| Keywords |
Anti-Bacterial Agents/analysis; Anti-Bacterial Agents/pharmacology; Bacterial Outer Membrane Proteins; Biological Assay/methods; Cell Membrane/metabolism; Cell Membrane/ultrastructure; Escherichia coli/cytology; Escherichia coli/enzymology; Escherichia coli/genetics; Inhibitory Concentration 50; Kinetics; N-Acetylglucosaminyltransferases/antagonists & inhibitors; N-Acetylglucosaminyltransferases/genetics; N-Acetylglucosaminyltransferases/metabolism; Peptidoglycan/biosynthesis; Reproducibility of Results; Substrate Specificity |
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