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Antimicrobial Agents and Chemotherapy, April 2005, p. 1410-1418, Vol. 49, No. 4
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.4.1410-1418.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Scintillation Proximity Assay for Inhibitors of Escherichia coli MurG and, Optionally, MraY

Sudha Ravishankar,{dagger} Vidya Prasanna Kumar,{dagger} B. Chandrakala, Ramesh K. Jha, Suresh M. Solapure, and Sunita M. de Sousa*

AstraZeneca India Pvt. Ltd., Bangalore, India

Received 12 July 2004/ Returned for modification 26 September 2004/ Accepted 27 December 2004

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 ß-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.

* Corresponding author. Mailing address: AstraZeneca India Pvt. Ltd., Hebbal, Bellary Rd., Bangalore 560 024, India. Phone: 91-80-2362-1212. Fax: 91-80-2362-1214. E-mail: sunita.desousa{at}astrazeneca.com.

{dagger} These authors contributed equally to the work presented here.

Antimicrobial Agents and Chemotherapy, April 2005, p. 1410-1418, Vol. 49, No. 4
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.4.1410-1418.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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