Purification, reconstitution, and inhibition of cytochrome P-450 sterol delta22-desaturase from the pathogenic fungus Candida glabrata.
Sterol delta22-desaturase has been purified from a strain of Candida glabrata with a disruption in the gene encoding sterol 14alpha-demethylase (cytochrome P-45051; CYP51). The purified cytochrome P-450 exhibited sterol delta22-desaturase activity in a reconstituted system with NADPH-cytochrome P-450 reductase in dilaurylphosphatidylcholine, with the enzyme kinetic studies revealing a Km for ergosta-5,7-dienol of 12.5 microM and a Vmax 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 delta22-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 delta22-desaturase to be an antifungal target and to contribute to the binding of drugs within the fungal cell.
Lamb
D C
DC
Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, Wales SY23 3DA, England S10 2UH, United Kingdom.
Maspahy
S
S
Kelly
D E
DE
Manning
N J
NJ
Geber
A
A
Bennett
J E
JE
Kelly
S L
SL
D000935:Antifungal Agents / Q000494:pharmacology; D002175:Candida / Q000201:enzymology*; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification*; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D029701:Saccharomyces cerevisiae Proteins
D000935:Antifungal Agents; D065607:Cytochrome P-450 Enzyme Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; C066577:ERG5 protein, S cerevisiae