P450
oxidoreductase (POR) has a pivotal role in facilitating electron transfer from
nicotinamide adenine dinucleotide phosphate to microsomal
cytochrome P450 (CYP)
enzymes, including the steroidogenic
enzymes CYP17A1 and CYP21A2. Mutations in POR have been shown recently to cause
congenital adrenal hyperplasia with apparent combined CYP17A1 and CYP21A2 deficiency that comprises a variable clinical phenotype, including
glucocorticoid deficiency,
ambiguous genitalia, and craniofacial malformations. To dissect structure-function relationships potentially explaining this phenotypic diversity, we investigated whether specific POR mutations have differential effects on CYP17A1 and CYP21A2. We compared the impact of missense mutations encoding for single
amino acid changes in three distinct regions of the POR molecule: 1), Y181D and H628P close to the central electron transfer area, 2) S244C located within the hinge close to the
flavin adenine dinucleotide and
flavin mononucleotide domains of POR, and 3) A287P that is clearly distant from the two other regions. Functional analysis using a yeast microsomal assay with coexpression of human CYP17A1 or CYP21A2 with wild-type or mutant human POR revealed equivalent decreases in CYP17A1 and CYP21A2 activities by Y181D, H628P, and S244C. In contrast, A287P had a differential inhibitory effect, with decreased catalytic efficiency (Vmax/Km) for CYP17A1, whereas CYP21A2 retained near normal activity. In vivo analysis of urinary
steroid excretion by gas chromatography/mass spectrometry in 11 patients with POR mutations showed that A287P homozygous patients had the highest
corticosterone/
cortisol metabolite ratios, further indicative of preferential inhibition of CYP17A1. These findings provide novel mechanistic insights into the redox regulation of human steroidogenesis. Differential interaction of POR with electron-accepting CYP
enzymes may explain the phenotypic variability in
POR deficiency, with additional implications for hepatic
drug metabolism by POR-dependant CYP
enzymes.