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Antimicrobial Agents and Chemotherapy, March 2004, p. 897-902, Vol. 48, No. 3
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.3.897-902.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Fluorescence Detection-Based Functional Assay for High-Throughput Screening for MraY

Aventis Pharma France, 93235 Romainville,¹ Enveloppes Bactériennes et Antibiotiques, UMR 8619 CNRS, Université de Paris-Sud, 91405 Orsay, France²

Received 31 July 2003/ Returned for modification 24 September 2003/ Accepted 20 November 2003

We have developed a novel assay specific to MraY, which catalyzes the first membrane step in the biosynthesis of bacterial cell wall peptidoglycan. This was accomplished by using UDP-MurNAc-N-dansylpentapeptide, a fluorescent derivative of the MraY nucleotide substrate, and a partially purified preparation of MraY solubilized from membranes of an Escherichia coli overproducing strain. Two versions of the assay were developed, one consisting of the high-pressure liquid chromatography separation of the substrate and product (dansylated lipid I) and the other, without separation and adapted to the high-throughput format, taking advantage of the different fluorescence properties of the nucleotide and lipid I in the reaction medium. The latter assay was validated with a set of natural and synthetic MraY inhibitors.

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