Open AccessNADPH Cytochrome P-450 Oxidoreductase and Susceptibility to Ketoconazole
Author(s) -
Venkateswarlu Kanamarlapudi,
Diane Kelly,
N. J. Manning,
Steven L. Kelly
Publication year - 1998
Publication title -
antimicrobial agents and chemotherapy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.07
H-Index - 259
eISSN - 1070-6283
pISSN - 0066-4804
DOI - 10.1128/aac.42.7.1756
Subject(s) - ketoconazole , ergosterol , complementation , biology , oxidoreductase , cytochrome , cytochrome p450 , biochemistry , strain (injury) , chemistry , microbiology and biotechnology , gene , phenotype , enzyme , antifungal , anatomy
The phenotype of a strain ofSaccharomyces 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 thecpr strain, but ketoconazole sensitivity increased 200-fold, indicating hypersensitivity to the alternative electron donor system incpr 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.