A multitarget assay for inhibitors of membrane-associated steps of peptidoglycan biosynthesis.
Anal Biochem
; 306(1): 17-22, 2002 Jul 01.
Article
em En
| MEDLINE
| ID: mdl-12069409
ABSTRACT
Peptidoglycan synthesis begins in the cytoplasm with the condensation of UDP-N-acetyl glucosamine (UDP-GlcNAc) and phosphoenolpyruvate catalyzed by UDP-N-acetylglucosamine enolpyruvoyl transferase. UDP-GlcNAc is also utilized as substrate for the glycosyltransferase MurG, a membrane-bound enzyme that catalyzes the production of lipid II. Membranes from Escherichia coli cells overproducing MurG support peptidoglycan formation at a rate approximately fivefold faster than membranes containing wild-type levels of MurG. Conditions have been optimized for the production of large amounts of membranes with increased levels of MurG, allowing the development of an assay suitable for high-throughput screening of large compound libraries. The quality of the purified membranes was assessed by electron microscopy and also by testing cross-linked peptidoglycan production. Moreover, kinetic studies allowed the determination of optimal concentrations of the substrates and membranes to be utilized for maximum sensitivity of the assay. Using a 96-well assay format, the IC50 values for vancomycin, tunicamycin, flavomycin, and bacitracin were 1.1 microM, 0.01 microg/ml, 0.03 microg/ml, and 0.7 microg/ml, respectively.
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Base de dados:
MEDLINE
Assunto principal:
Proteínas da Membrana Bacteriana Externa
/
Bioensaio
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Peptidoglicano
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Membrana Celular
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N-Acetilglucosaminiltransferases
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Antibacterianos
Idioma:
En
Ano de publicação:
2002
Tipo de documento:
Article
País de afiliação:
Estados Unidos