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

7 Citations (Scopus)

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
StatePublished - 1997
Externally publishedYes

Fingerprint

Glycopeptides
Assays
Screening
Enterococcus faecium
Genes
Vancomycin
Ligation
Lactic Acid
Depsipeptides
Ninhydrin
Teicoplanin
Peptidoglycan
Dipeptides
Ligases
Cadmium
Cell Wall
Hydrolysis
Phosphates
Escherichia coli
Anti-Bacterial Agents

ASJC Scopus subject areas

  • Biotechnology
  • Clinical Biochemistry
  • Analytical Chemistry

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.

Development of a coupled VanA/VanX assay : Screening for inhibitors of glycopeptide resistance. / Pratt, Steven D.; Xuei, Xiaoling; Mackinnon, Alexander C.; Nilius, Angela M.; Hensey-Rudloff, Dena M.; Zhong, Ping; Katz, Leonard.

In: Journal of Biomolecular Screening, Vol. 2, No. 4, 1997, p. 241-247.

Research output: Contribution to journalArticle

Pratt, SD, Xuei, X, Mackinnon, AC, Nilius, AM, Hensey-Rudloff, DM, Zhong, P & Katz, L 1997, 'Development of a coupled VanA/VanX assay: Screening for inhibitors of glycopeptide resistance', Journal of Biomolecular Screening, vol. 2, no. 4, pp. 241-247.
Pratt SD, Xuei X, Mackinnon AC, Nilius AM, Hensey-Rudloff DM, Zhong P et al. Development of a coupled VanA/VanX assay: Screening for inhibitors of glycopeptide resistance. Journal of Biomolecular Screening. 1997;2(4):241-247.
Pratt, Steven D. ; Xuei, Xiaoling ; Mackinnon, Alexander C. ; Nilius, Angela M. ; Hensey-Rudloff, Dena M. ; Zhong, Ping ; Katz, Leonard. / Development of a coupled VanA/VanX assay : Screening for inhibitors of glycopeptide resistance. In: Journal of Biomolecular Screening. 1997 ; Vol. 2, No. 4. pp. 241-247.
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