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

Abstract

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 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.

Arabidopsis CYP735A1 and CYP735A2 Encode Cytokinin Hydroxylases That Catalyze the Biosynthesis of trans-Zeatin

“…The microsomal fraction was prepared by the method of Venkateswarlu et al (21), except that the buffer A did not contain reduced glutathione. After ultracentrifugation, the pellet was further washed once with the buffer A.…”

Biodiversity of the P450 catalytic cycle: yeast cytochrome b5/NADH cytochrome b5 reductase complex efficiently drives the entire sterol 14‐demethylation (CYP51) reaction

“…Catalytic activity studies on sterol 14K-demethylase with cytochrome b 5 , NADH cytochrome b 5 reductase and NADPH cytochrome P450 reductase (CPR) Previously, we have reported that disruption of S. cerevisiae NADPH cytochrome P450 reductase (CPR) gene resulted in a strain accumulating ergosterol [19]. This indicated that the cytochrome P450 sterol biosynthetic enzymes, namely sterol 14K-demethylase (CYP51) and sterol v 22 -desaturase (CYP61), could receive electrons to utilise in their catalytic reaction mechanisms from other electron donor proteins other than CPR.…”

“…Expression and puri¢cation of recombinant S. cerevisiae cytochrome P450 reductase S. cerevisiae JL20 transformants carrying native yeast CPR expression vector was grown in yeast minimal medium containing Difco yeast nitrogen base without amino acids (1.34%, w/v), 100 mg/l Lhistidine and glucose (2%, w/v) at 30³C until glucose was completely consumed and then heterologous expression was induced with galactose (3%, w/v) for 20 h [19,26]. Cells were harvested by centrifugation and subjected to mechanical breakage.…”

Liver microsomal lipid enhances the activity and redox coupling of colocalized cytochrome P450 reductase‐cytochrome P450 3A4 in nanodiscs

“…Taken together, our results are a direct demonstration of the requirement for coinsertion of CPR and CYP3A4 within the same phospholipid membrane for reconstitution of activity. Interestingly, yeast CPR lacking the N‐terminal MAD does support CYP‐dependent sterol biosynthesis, both in vivo and in a reconstituted assay 53. The explanation for this apparent qualitative difference between yeast and mammalian CPR is not known.…”