Purification, Reconstitution, and Inhibition of Cytochrome P-450 Sterol Δ 22 -Desaturase from the Pathogenic Fungus Candida glabrata

Abstract

ABSTRACT Sterol Δ 22 -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 Δ 22 -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 Δ 22 -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 Δ 22 -desaturase to be an antifungal target and to contribute to the binding of drugs within the fungal cell.