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. Electron transfer in POR occurs from NADH to FAD to FMN, and the flexible hinge region in POR is essential for domain movements to bring the FAD and FMN close together for electron transfer. We tested the effect of variations in the hinge region of POR to check if the effects would be similar across all redox partners or there will be differences in activities. Here we are reporting the effects of a POR genetic variant P284T located in the hinge region of POR that is necessary for the domain movements and internal electron transfer between co-factors. Human wild-type and P284T mutant of POR and cytochrome P450 proteins were expressed in bacteria, purified, and reconstituted for enzyme assays. 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, indicating intact direct electron transfer from FAD to ferricyanide, 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. The negative impact of P284T mutation in the hinge region of POR seems to be due to disruption of FAD to FMN electron transfer. These results further emphasize the importance of hinge region in POR for protein flexibility and electron transfer within POR as well as the interaction of POR with different redox partners.
Highlights
The NADPH cytochrome P450 reductase (POR, NCBI# NP_000932, UniProt# P16435) is required for the enzymatic activities for all human cytochromes P450 proteins located in the endoplasmic reticulum (Pandey and Flück, 2013; Flück and Pandey, 2019)
We have investigated the effects of P450 oxidoreductase (POR) variant P284T located near the hinge region of POR that is crucial for interactions with redox partner proteins, including P450s, to transfer electrons from NADPH
The loss of activities with cytochrome c and MTT indicates disruption of electron transport from flavin adenine dinucleotide (FAD) to flavin mononucleotide (FMN), which could be due to conformational instability due to a P284T mutation affecting domain movements as well as a chain reaction starting from the disruption of electron transfer from NADPH to FAD
Summary
The NADPH cytochrome P450 reductase (POR, NCBI# NP_000932, UniProt# P16435) is required for the enzymatic activities for all human cytochromes P450 proteins located in the endoplasmic reticulum (Pandey and Flück, 2013; Flück and Pandey, 2019). The type 1 P450s are located in the mitochondria and use the adrenodoxin/adrenodoxin reductase system as their redox partner (Omura, 2006; Zalewski et al, 2016). The type 2 cytochrome P450s are located in the endoplasmic reticulum and use a single diflavin reductase, POR, as their redox partner (Lu et al, 1969; Masters, 2005).
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