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

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 -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.The biosynthesis of bacterial peptidoglycan is a complex two-stage process. The first stage consists in the formation of the monomer unit GlcNAc-MurNAc-(pentapeptide), whereas the second one concerns polymerization and maturation reactions (28). In the first stage, two enzymes, MraY and MurG, have in common their attachment to the cytoplasmic membrane and their processing of lipid intermediates: MraY transfers the phospho-MurNAc-pentapeptide motif onto lipid carrier undecaprenyl phosphate, yielding MurNAc-(pentapeptide)-pyrophosphoryl undecaprenol (lipid I), and MurG then adds the GlcNAc residue to yield GlcNAc-MurNAc-(pentapeptide)-pyrophosphoryl undecaprenol (lipid II), which contains the whole monomer unit. In the present study, we have focused our attention to the MraY transferase, which is an integral membrane protein with ten transmembrane segments, five cytoplasmic domains, and six periplasmic domains including the N-and C-terminal ends (4). Besides the synthesis of lipid I, which is fully reversible, MraY catalyzes in vitro an exchange reaction between UMP and UDP-MurNAc-pentapeptide (23). Owing to its exclusive presence in bacteria and to its essential character, which was demonstrated in Escherichia coli (5), MraY is a target of interest for the discovery of novel antibacterial agents. It is inhibited by non-clinically used antibiotics such as tunicamycin, amphomycin, mureidomycin, liposidomycin, and muraymycins (7,9,22). Recently, simplified analogues of liposidomycin, named riburamycins, have been shown to be powerful MraY inhibitors and to possess antibacterial activities against gram-positive organisms (13-16) (J. Biton, K. Braham, C. Dini, O. Krebs, P. Lassaigne, F. Monti, T. Stachyra, V. Steier, and J. Zhang, Abstr. 42nd Intersci. Conf. Antimicrob. Agents Chemother., abstr. F-361, 2002). To date, several specific assays for the MraY activity have been published (6,17,18,27,29,30); however, very few can be adapted to the high-throughput format. In the present work, we took advantage of the fluorescent properties of UDP-MurNAc-N ε -dansylpentapeptide and dansylated lipid I (6, 29) to set up a high-throughput screen (HTS) assay for MraY, which was validated with a set of natural and synthetic ...

Preparative Scale Cell-free Production and Quality Optimization of MraY Homologues in Different Expression Modes

“…The difficult production of MraY and other MPs involved in peptidoglycan biosynthesis so far have largely retarded the detailed study of their biosynthetic mechanisms and the search of novel antibacterials. Nevertheless, the substrate specificity of MraY was intensively studied and a number of enzymatic assays designed for throughput screening of inhibitors have been developed (31)(32)(33).…”

A High-Throughput, Homogeneous, Fluorescence Resonance Energy Transfer-Based Assay for Phospho-N-acetylmuramoyl-pentapeptide Translocase (MraY)

“…Solapure et al 8 reported an IC 50 of 360 nM in an assay containing 9 µM UNAM-pp, 0.1% Triton X-100, 25 mM Mg 2+ , and only endogenous C 55 P from the membranes used as a source of MraY. Stachyra et al 7 reported an IC 50 of 2 µM in an assay containing 50 µM C 55 P and 25 µM dansyl-UNAM-pp in 0.1% TX-100 and 50 mM Mg . Several factors may contribute to the observed differences, including differences in the substrate, detergent, Mg…”

“…The column was eluted with 100%A for 3 min, a gradient of 100%A to 100%B in 2 min, then 100%B for 5 min. 7 The flow rate was 0.5 mL/min at room temperature. The B-UNAM-pp peak eluted at 5.7 min.…”

Assessing HTS Performance Using BioAssay Ontology: Screening and Analysis of a Bacterial Phospho-N-Acetylmuramoyl-Pentapeptide Translocase Campaign

“…10 IC 50 values reported using other assays in the range of 360 nM to 2 mM. 8,11,12 Z 0 values for the 1,536-well assay during the HTS were equal to or greater than 0.6 and the signal-to-background ratio was 1.4-1.6. Primary MraY HTS and associated confirmatory assays were screened using the three compound libraries described in the ''Materials and Methods'' section.…”