Development of a coupled VanA/VanX assay: Screening for inhibitors of glycopeptide resistance

Steven D. Pratt, Xiaoling Xuei, Alexander C. Mackinnon, Angela M. Nilius, Dena M. Hensey-Rudloff, Ping Zhong, Leonard Katz

Research output: Contribution to journalArticle

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Abstract

Resistance in Enterococcus faecium to the glycopeptide antibiotics vancomycin and teicoplanin is encoded by five genes: vanR, vanS, vanH, vanA, and vanX.1 The mechanism of resistance involves replacement of the dipeptide D-Ala-D-Ala, destined for the peptidoglycan layer with the depsipeptide D-Ala-D-lactate. This alteration lowers the binding affinity of vancomycin for the bacterial cell wall by a factor of 1000. The functions of VanA and VanX are the ligation of D-Ala and D-lactate, and the hydrolysis of D-Ala-D-Ala, respectively. We report here the overexpression of both genes as well as the D-Ala-D-Ala ligase (Ddl) from Enterococcus faecium, development of a coupled assay and several inhibitors obtained by high-throughput screening (HTS). All genes were expressed in E. coli by translational coupling to kdsB, the CMP-KDO synthetase gene, under control of a modified lac promoter. The coupled VanA/VanX assay employs colorimetric detection of inorganic phosphate (Pi) released in the VanA ligation reaction, with the VanX dipeptidase activity providing the D-Ala substrate for VanA. A secondary VanX assay uses cadmium-ninhydrin colorimetric detection of free amino acid released by the dipeptidase activity of the enzyme on D-Ala-D-Ala. We have also developed an assay using Ddl ligase. Over 250,000 compounds have been screened to date using the coupled assay.

Original languageEnglish (US)
Pages (from-to)241-247
Number of pages7
JournalJournal of Biomolecular Screening
Volume2
Issue number4
DOIs
StatePublished - Jan 1 1997

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ASJC Scopus subject areas

  • Analytical Chemistry
  • Biotechnology
  • Biochemistry
  • Molecular Medicine
  • Pharmacology
  • Drug Discovery

Cite this

Pratt, S. D., Xuei, X., Mackinnon, A. C., Nilius, A. M., Hensey-Rudloff, D. M., Zhong, P., & Katz, L. (1997). Development of a coupled VanA/VanX assay: Screening for inhibitors of glycopeptide resistance. Journal of Biomolecular Screening, 2(4), 241-247. https://doi.org/10.1177/108705719700200409