title: Scintillation proximity assay for inhibitors of Escherichia coli MurG and, optionally, MraY.
abstract: MurG and MraY, essential enzymes involved in the synthesis of bacterial peptidoglycan, are difficult to assay because the substrates are lipidic and hard to prepare in large quantities. Based on the use of Escherichia coli membranes lacking PBP1b, we report a high-throughput method to measure the activity of MurG and, optionally, MraY as well. In these membranes, incubation with the two peptidoglycan sugar precursors results in accumulation of lipid II rather than the peptidoglycan produced by wild-type membranes. MurG was assayed by addition of UDP-[3H]N-acetylglucosamine to membranes in which lipid I was preformed by incubation with UDP-N-acetyl-muramylpentapeptide, and the product was captured by wheat germ agglutinin scintillation proximity assay beads. In a modification of the assay, the activity of MraY was coupled to that of MurG by addition of both sugar precursors together in a single step. This allows simultaneous detection of inhibitors of either enzyme. Both assays could be performed using wild-type membranes by addition of the transglycosylase inhibitor moenomycin. Nisin and vancomycin inhibited the MurG reaction; the MraY-MurG assay was inhibited by tunicamycin as well. Inhibitors of other enzymes of peptidoglycan synthesis--penicillin G, moenomycin, and bacitracin--had no effect. Surprisingly, however, the beta-lactam cephalosporin C inhibited both the MurG and MraY-MurG assays, indicating a secondary mechanism by which this drug inhibits bacterial growth. In addition, it inhibited NADH dehydrogenase in membranes, a hitherto-unreported activity. These assays can be used to screen for novel antibacterial agents.
authors.affiliation: AstraZeneca India Pvt. Ltd., Hebbal, Bellary Rd., Bangalore 560 024, India.
authors.forename: Sudha
authors.initials: S
authors.lastname: Ravishankar
authors.forename: Vidya Prasanna
authors.initials: VP
authors.lastname: Kumar
authors.forename: B
authors.initials: B
authors.lastname: Chandrakala
authors.forename: Ramesh K
authors.initials: RK
authors.lastname: Jha
authors.forename: Suresh M
authors.initials: SM
authors.lastname: Solapure
authors.forename: Sunita M
authors.initials: SM
authors.lastname: de Sousa
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001425:Bacterial Outer Membrane Proteins / Q000037:antagonists & inhibitors*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002511:Cephalosporins / Q000494:pharmacology; D002854:Chromatography, Paper; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000235:genetics; D008826:Microbial Sensitivity Tests / Q000379:methods; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000037:antagonists & inhibitors*; D046915:Penicillin-Binding Proteins / Q000235:genetics; D010457:Peptidoglycan / Q000096:biosynthesis; D046928:Peptidoglycan Glycosyltransferase / Q000235:genetics; D012588:Scintillation Counting; D012680:Sensitivity and Specificity; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase / Q000235:genetics; D014166:Transferases / Q000037:antagonists & inhibitors*; D017855:Transferases (Other Substituted Phosphate Groups); D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives* / Q000096:biosynthesis
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002511:Cephalosporins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D046915:Penicillin-Binding Proteins; D010457:Peptidoglycan; D014538:Uridine Diphosphate N-Acetylmuramic Acid; C069249:muramyl-NAc-(pentapeptide)pyrophosphoryl-undecaprenol; C025163:cephalosporin C; D014166:Transferases; D017351:N-Acetylglucosaminyltransferases; D046928:Peptidoglycan Glycosyltransferase; C478681:penicillin-binding protein 1B, E coli; C064057:UDP-N-acetylglucosamine-N-acetylmuramyl-(pentapeptide)pyrophosphoryl-undecaprenol N-acetylglucosamine transferase; D017855:Transferases (Other Substituted Phosphate Groups); C079803:mraY protein, Bacteria; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase