Loss of multiple enzyme activities due to the human genetic variation P284T in NADPH cytochrome P450 oxidoreductase

Abstract

Cytochromes P450 located in the endoplasmic reticulum require NADPH cytochrome P450 oxidoreductase (POR) for their catalytic activities. Mutations in POR cause multiple disorders in humans related to the biosynthesis of steroid hormones and also affect drug-metabolizing cytochrome P450 activities. Here we are reporting the effects of a POR genetic variant P284T which is located in the hinge region of POR that is necessary for the flexibility of domain movements. Human wild-type and P284T mutant of POR, as well as cytochrome P450 proteins, were expressed in bacteria, purified and then reconstituted in liposomes for enzyme kinetic assays. Quality of POR proteins was checked by cytochrome c, ferricyanide and tetrazolium dye reduction assay and measurements flavin content. We found that for the P284T variant of POR the cytochrome c reduction activity was reduced to 47% of the WT and MTT reduction was reduced to only 15% of the WT. No impact on ferricyanide reduction activity was observed, but a severe loss of CYP19A1 (aromatase) activity was observed (9% of WT). In the assays of drug metabolizing cytochrome P450 enzymes, the P284T variant of POR showed 26% activity for CYP2C9, 44% activity for CYP2C19, 23% activity for CYP3A4 and 44% activity in CYP3A5 assays compared to the WT POR. These results indicate a severe effect on several cytochrome P450 activities due to the P284T variation in POR which suggests a negative impact on both the steroid as well as drug metabolism in the individuals carrying this variation.