title: Characterization of Saccharomyces cerevisiae CYP61, sterol delta22-desaturase, and inhibition by azole antifungal agents.
abstract: Cytochrome P-45061 (CYP61) was a cytochrome P-450 revealed during the yeast genome project when chromosome XIII was sequenced. Here we report on the properties of this second microsomal P-450 of vegetatively growing yeast. The enzyme kinetics associated with its endogenous role in sterol Delta22-desaturation revealed a Km of 20.4 microM and a Vmax of 2.9nmol/min/nmol CYP61. The affinity of the enzyme for antifungal drugs was characterized to investigate its potential role in determining tolerance to these sterol 14alpha-demethylase (CYP51) inhibitors. Drug binding induced a type II spectral change, which became saturated at equimolar concentrations of azole drug and P-450. Fluconazole exhibited slightly reduced affinity in comparison to ketoconazole as indicated by carbon monoxide displacement. These and Ki determination for fluconazole (0.14 nM) revealed CYP61 to have a similar affinity to azole drugs when compared with data available for CYP51, and the implications for antifungal treatment were considered.
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, Sheffield University, Sheffield, S10 2UH, United Kingdom. S.Kelly@sheffield.ac.uk
authors.forename: S L
authors.initials: SL
authors.lastname: Kelly
authors.forename: D C
authors.initials: DC
authors.lastname: Lamb
authors.forename: B C
authors.initials: BC
authors.lastname: Baldwin
authors.forename: A J
authors.initials: AJ
authors.lastname: Corran
authors.forename: D E
authors.initials: DE
authors.lastname: Kelly
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D004875:Ergosterol / Q000031:analogs & derivatives / Q000378:metabolism; D015725:Fluconazole / Q000494:pharmacology*; D007654:Ketoconazole / Q000494:pharmacology*; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013054:Spectrophotometry, Atomic; D058886:Sterol 14-Demethylase
chemical_list: D000935:Antifungal Agents; D065607:Cytochrome P-450 Enzyme Inhibitors; D029701:Saccharomyces cerevisiae Proteins; C012287:22,23-dihydroergosterol; D015725:Fluconazole; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; C066577:ERG5 protein, S cerevisiae; D058886:Sterol 14-Demethylase; D007654:Ketoconazole; D004875:Ergosterol