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.
Stachyra
Thérèse
T
Aventis Pharma France, 93235 Romainville, France.
Dini
Christophe
C
Ferrari
Paul
P
Bouhss
Ahmed
A
van Heijenoort
Jean
J
Mengin-Lecreulx
Dominique
D
Blanot
Didier
D
Biton
Jacques
J
Le Beller
Dominique
D
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001419:Bacteria / Q000187:drug effects / Q000235:genetics; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors*; D002851:Chromatography, High Pressure Liquid; D003619:Dansyl Compounds / Q000737:chemistry / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology; D008826:Microbial Sensitivity Tests; D009005:Monosaccharides / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology*; D010957:Plasmids / Q000187:drug effects / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014166:Transferases / Q000037:antagonists & inhibitors*; D017855:Transferases (Other Substituted Phosphate Groups)
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D003619:Dansyl Compounds; D009005:Monosaccharides; D009842:Oligopeptides; C483961:UDP-MurNAc-N(epsilon)-dansylpentapeptide; C438940:lipid I; D014166:Transferases; D017855:Transferases (Other Substituted Phosphate Groups); C079803:mraY protein, Bacteria