The N-Terminal Membrane Domain of Yeast NADPH-Cytochrome P450 (CYP) Oxidoreductase Is Not Required for Catalytic Activity in Sterol Biosynthesis or in Reconstitution of CYP Activity

Diane E. Kelly,David C. Lamb,K. Venkateswarlu,Steven L. Kelly,Nigel J. Manning

doi:10.1074/jbc.273.8.4492

Diane E. Kelly, David C. Lamb + Show 3 more

Open Access

https://doi.org/10.1074/jbc.273.8.4492

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Abstract

The disruption of Saccharomyces cerevisiae NADPH- cytochrome P450 oxidoreductase (CPR) gene resulted in a viable strain accumulating approximately 25% of the ergosterol observed in a sterol wild-type parent. The associated phenotypes could be reversed in transformants after expression of native CPR and a mutant lacking the N-terminal 33 amino acids, which localized in the cytosol. This indicated availability of the CPR in each case to function with the monooxygenases squalene epoxidase, CYP51, and CYP61 in the ergosterol biosynthesis pathway. Purification of the cytosolic mutant CPR indicated properties identical to native CPR and an ability to reconstitute ergosterol biosynthesis when added to a cell-free system, as well as to allow reconstitution of activity with purified CYP61, sterol 22-desaturase. This was also observed for purified Candida albicans and human CYP51 in reconstituted systems. The ability of the yeast enzyme to function in a soluble form differed from human CPR, which is shown to be inactive in reconstituting CYP activity.

Highlights

The disruption of Saccharomyces cerevisiae NADPHcytochrome P450 oxidoreductase (CPR) gene resulted in a viable strain accumulating approximately 25% of the ergosterol observed in a sterol wild-type parent
The ability of the yeast enzyme to function in a soluble form differed from human CPR, which is shown to be inactive in reconstituting cytochrome P450 (CYP) activity
We are interested in the yeast CPR, and we developed and characterized systems to examine the function of the soluble domain of the enzyme with comparison to human CPR

Summary

EXPERIMENTAL PROCEDURES

Strains—Escherichia coli strain DH5␣ was used for cloning, and Saccharomyces cerevisiae strain JL20 (MAT a, leu 112, his519, ade100, ura3-52) was used for expression and gene disruption. Chromosomal CPR in the haploid JL20 yeast strain was inactivated by transplacement using the SalIHindIII fragment containing CPR::URA3, and the disruption was confirmed by PCR, using the cells of URA3 colonies of the transformed JL20 strain, the primers and conditions mentioned above (the cells were heated in a microwave for 30 s and quickly cooled on ice for lysis before being subjected to PCR), and ketoconazole susceptibility tests. Reconstitution of CYP Activity—The standard reaction mixtures contained purified S. cerevisiae CYP61, C. albicans and human CYP51 (0.5 nmol), 1 unit of either native yeast and human CPR or the N-terminal truncated soluble CPR forms, 13 nmol of the respective substrate (ergosta-5,7-dienol for S. cerevisiae CYP61 and dihydrolanosterol or 24methylene-24, 25-dihydrolanosterol for human and C. albicans CYP51, respectively) dispersed in 80 nmol dilauroylphosphatidylcholine; the reaction volume was adjusted to 950 ␮l with 100 mM potassium phosphate buffer, pH 7.2.

Ergosterol Dry weight of cells Total sterols

RESULTS

Per mg of protein

In vitro ergosterol biosynthesis

DISCUSSION