Purification, reconstitution, and inhibition of cytochrome P-450 sterol Δ22-desaturase from the pathogenic fungus Candida glabrata

David C. Lamb, Segula Maspahy, Diane E. Kelly, Nigel J. Manning, Antonia Geber, John E. Bennett, Steven L. Kelly

Research output: Contribution to journalArticle

Abstract

Sterol Δ22-desaturase has been purified from a strain of Candida glabrata with a disruption in the gene encoding sterol 14α-demethylase (cytochrome P-45051; CYP51). The purified cytochrome P-450 exhibited sterol Δ22-desaturase activity in a reconstituted system with NADPH-cytochrome P- 450 reductase in dilaurylphosphatidylcholine, with the enzyme kinetic studies revealing a K(m) for ergosta-5,7-dienol of 12.5 μM and a V(max) of 0.59 nmol of this substrate metabolized/min/nmol of P-450. This enzyme is encoded by CYP61 (ERG5) in Saccharomyces cerevisiae, and homologues have been shown in the Candida albicans and Schizosaccharomyces pombe genome projects. Ketoconazole, itraconazole, and fluconazole formed low-spin complexes with the ferric cytochrome and exhibited type II spectra, which are indicative of an interaction between the azole moiety and the cytochrome heme. The azole antifungal compounds inhibited reconstituted sterol Δ22-desaturase activity by binding to the cytochrome with a one-to-one stoichiometry, with total inhibition of enzyme activity occurring when equimolar amounts of azole and cytochrome P-450 were added. These results reveal the potential for sterol Δ22-desaturase to be an antifungal target and to contribute to the binding of drugs within the fungal cell.

Original languageEnglish (US)
Pages (from-to)1725-1728
Number of pages4
JournalAntimicrobial agents and chemotherapy
Volume43
Issue number7
DOIs
StatePublished - Jul 1999

ASJC Scopus subject areas

  • Pharmacology
  • Pharmacology (medical)
  • Infectious Diseases

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