NADPH cytochrome P-450 oxidoreductase and susceptibility to ketoconazole.
The phenotype of a strain of Saccharomyces cerevisiae containing a disruption of the gene encoding NADPH cytochrome P-450 oxidoreductase (CPR) was quantified biochemically and microbiologically, as were those of various transformants of this strain after expression of native CPR, cytochrome P-45051 (CYP51), and a fusion protein of CYP51-CPR (FUS). Only a 4-fold decrease in ergosterol biosynthesis was observed for the cpr strain, but ketoconazole sensitivity increased 200-fold, indicating hypersensitivity to the alternative electron donor system in cpr strains. Both phenotypes could be reversed in transformants expressing the CPR and FUS, indicating the availability of the CPR in FUS as well as the expressed native CPR for monoxygenase-associated reactions. The complementation of function was observed both in vitro and in vivo for the monoxygenases squalene epoxidase, CYP51, and CYP61 in the ergosterol biosynthesis pathway with which CPR is coupled. Overexpression of CYP51 and FUS produced different levels of ketoconazole resistance in wild-type cells, indicating that the availability of CPR may limit the potential of overproduction of CYP51 as a mechanism of resistance to azole antifungal agents.
Venkateswarlu
K
K
Institute of Biological Sciences, University of Wales Aberystwyth, Ceredigion, United Kingdom.
Kelly
D E
DE
Manning
N J
NJ
Kelly
S L
SL
D000935:Antifungal Agents / Q000378:metabolism / Q000494:pharmacology*; D002384:Catalysis; D002461:Cell Line, Transformed; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005816:Genetic Complementation Test; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D007654:Ketoconazole / Q000378:metabolism / Q000494:pharmacology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism; D034702:RNA-Binding Protein FUS; D012261:Ribonucleoproteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000502:physiology; D058886:Sterol 14-Demethylase
D000935:Antifungal Agents; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D034702:RNA-Binding Protein FUS; D012261:Ribonucleoproteins; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase; D009251:NADPH-Ferrihemoprotein Reductase; D007654:Ketoconazole