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.

Differential effects of variations in human P450 oxidoreductase on the aromatase activity of CYP19A1 polymorphisms R264C and R264H

“…Huang et al have reported a 54% loss of CYP17A1 17α-hydroxylase activity with POR P284L but found the 17,20 lyase activity of CYP17A1 to be similar to the wild type POR [76]. Recently we have shown that the P284L and P284T variants of POR showed reduced enzymatic activities with several drug-metabolizing cytochromes P450 [83,87]. There was a 55% loss of activity in CYP2C19 and CYP3A5 assays, while a 78% loss of activity was observed in CYP2C9 assays with the P284L variant of POR compared to the WT protein [87].…”

“…In case of P284T variant of POR a reduction of both the FAD as well as FMN content has been observed [83]. The drug metabolizing cytochrome P450 enzyme activities were also reduced when the POR-P284T was used as a redox partner (CYP2C9, 26%; CYP2C19, 44%; CYP3A4, 23% and CYP3A5, 44% of WT POR) [83]. Because the flexible hinge region of POR is necessary for the movement of domains that brings the FAD closer to the FMN for the transfer of electrons, a change in the hinge region may alter the electron transfer process in POR, affecting the activities of redox partners [92].…”

“…Flavin content was not affected by the variation P284L in POR [64], indicating a change in protein-protein interactions, both the inter as well as intramolecular; may be behind the reduced CYP19A1 activities. In case of P284T variant of POR a reduction of both the FAD as well as FMN content has been observed [83]. The drug metabolizing cytochrome P450 enzyme activities were also reduced when the POR-P284T was used as a redox partner (CYP2C9, 26%; CYP2C19, 44%; CYP3A4, 23% and CYP3A5, 44% of WT POR) [83].…”

“…The P284L is a rare variant found mostly in Europeans, and the P284T variant had been only observed in an African population [64,82,83]. The Y607C is a rare genetic variant found in both the Asian and European populations ( Table 2).…”