Mechanism and inhibition of the FabI enoyl-ACP reductase from Burkholderia pseudomallei

Nina Liu, Jason E. Cummings, Kathleen England, Richard A. Slayden, Peter J. Tonge

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

Objectives: As an initial step in developing novel antibacterials against Burkholderia pseudomallei, we have characterized the FabI enoyl-ACP reductase homologues in the type II fatty acid biosynthesis pathway from this organism and performed an initial enzyme inhibition study. Methods: A BLAST analysis identified two FabI enoyl-ACP reductase homologues, bpmFabI-1 and bpmFabI-2, in the B. pseudomallei genome, which were cloned, overexpressed in Escherichia coli and purified. Steady-state kinetics was used to determine the reaction mechanism and the sensitivity of bpmFabI-1 to four diphenyl ether FabI inhibitors. The antibacterial activity of the inhibitors was assessed using a wild-type strain of Burkholderia thailandensis (E264) and an efflux pump mutant (Bt38). Results: Consistent with its annotation as an enoyl-ACP reductase, bpmFabI-1 catalysed the NADH-dependent reduction of 2-trans-dodecenoyl-CoA via a sequential Bi Bi mechanism. In contrast, bpmFabI-2 was inactive with all substrates tested and only bpmfabI-1 was transcriptionally active under the growth conditions employed. The sensitivity of bpmFabI-1 to four diphenyl ethers was evaluated and in each case the compounds were slow-onset inhibitors with Ki values of 0.5-2 nM. In addition, triclosan and PT01 had MIC values of 30 and 70 mg/L for B. pseudomallei as well as a wild-type strain of B. thailandensis (E264), but MIC values of <1 mg/L for the efflux pump mutant Bt38. A reduction in MIC values was also observed for the pump mutant strain with the other diphenyl ethers. Conclusions: Provided that efflux can be circumvented, bpmFabI-1 is a suitable target for drug discovery.

Original languageEnglish (US)
Article numberdkq509
Pages (from-to)564-573
Number of pages10
JournalJournal of Antimicrobial Chemotherapy
Volume66
Issue number3
DOIs
StatePublished - Mar 1 2011

Keywords

  • B. pseudomallei
  • Efflux pumps
  • Slow-onset inhibition

ASJC Scopus subject areas

  • Pharmacology
  • Microbiology (medical)
  • Infectious Diseases
  • Pharmacology (medical)

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    Liu, N., Cummings, J. E., England, K., Slayden, R. A., & Tonge, P. J. (2011). Mechanism and inhibition of the FabI enoyl-ACP reductase from Burkholderia pseudomallei. Journal of Antimicrobial Chemotherapy, 66(3), 564-573. [dkq509]. https://doi.org/10.1093/jac/dkq509