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

Thérèse Stachyra·Christophe Dini·Paul Ferrari·Ahmed Bouhss·Jean van Heijenoort·Dominique Mengin-Lecreulx·Didier Blanot·Jacques Biton·Dominique Le Beller
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Abstract

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.

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Anti-Bacterial AgentsBacteriaBacterial ProteinsChromatography, High Pressure LiquidDansyl CompoundsDrug Evaluation, PreclinicalEscherichia coliMicrobial Sensitivity TestsMonosaccharidesOligopeptidesPlasmidsSpectrometry, FluorescenceSpectrophotometry, UltravioletTransferasesTransferases (Other Substituted Phosphate Groups)Journal ArticleResearch Support, Non-U.S. Gov't

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Thérèse Stachyra, Christophe Dini, Paul Ferrari, Ahmed Bouhss, Jean van Heijenoort, Dominique Mengin-Lecreulx, Didier Blanot, Jacques Biton, Dominique Le Beller. 2004. Fluorescence detection-based functional assay for high-throughput screening for MraY.. https://doi.org/10.1128/aac.48.3.897-902.2004

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