Fluorescence detection-based functional assay for high-throughput screening for MraY

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(epsilon)-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.