Fluorescence detection-based functional assay for high-throughput screening for MraY.
Antimicrob Agents Chemother
; 48(3): 897-902, 2004 Mar.
Article
em En
| MEDLINE
| ID: mdl-14982781
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Oligopeptídeos
/
Proteínas de Bactérias
/
Transferases
/
Compostos de Dansil
/
Antibacterianos
Tipo de estudo:
Diagnostic_studies
/
Screening_studies
Idioma:
En
Ano de publicação:
2004
Tipo de documento:
Article