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Antimicrobial Agents and Chemotherapy, March 2004, p. 897-902, Vol. 48, No. 3
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.3.897-902.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

Thérèse Stachyra,1 Christophe Dini,1 Paul Ferrari,1 Ahmed Bouhss,2 Jean van Heijenoort,2 Dominique Mengin-Lecreulx,2 Didier Blanot,2* Jacques Biton,1 and Dominique Le Beller1

Aventis Pharma France, 93235 Romainville,1 Enveloppes Bactériennes et Antibiotiques, UMR 8619 CNRS, Université de Paris-Sud, 91405 Orsay, France2

Received 31 July 2003/ Returned for modification 24 September 2003/ Accepted 20 November 2003

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{varepsilon}-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.

* Corresponding author. Mailing address: Enveloppes Bactériennes et Antibiotiques, UMR 8619 CNRS, Université de Paris-Sud, BÂtiment 430, 91405 Orsay, France. Phone: 33-1-69-15-61-34. Fax: 33-1-69-85-37-15. E-mail: didier.blanot{at}ebp.u-psud.fr.

Antimicrobial Agents and Chemotherapy, March 2004, p. 897-902, Vol. 48, No. 3
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.3.897-902.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • van Heijenoort, J. (2007). Lipid Intermediates in the Biosynthesis of Bacterial Peptidoglycan. Microbiol. Mol. Biol. Rev. 71: 620-635 [Abstract] [Full Text]  
  • Price, N. P., Momany, F. A. (2005). Modeling bacterial UDP-HexNAc: polyprenol-P HexNAc-1-P transferases. Glycobiology 15: 29R-42R [Abstract] [Full Text]  
  • Bouhss, A., Crouvoisier, M., Blanot, D., Mengin-Lecreulx, D. (2004). Purification and Characterization of the Bacterial MraY Translocase Catalyzing the First Membrane Step of Peptidoglycan Biosynthesis. J. Biol. Chem. 279: 29974-29980 [Abstract] [Full Text]  


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