Variability in Loss of Multiple Enzyme Activities Due to the Human Genetic Variation P284T Located in the Flexible Hinge Region of NADPH Cytochrome P450 Oxidoreductase.

Parween, Shaheena; Rojas Velazquez, Maria Natalia; Udhane, Sameer S.; Kagawa, Norio; Pandey, Amit V. (2019). Variability in Loss of Multiple Enzyme Activities Due to the Human Genetic Variation P284T Located in the Flexible Hinge Region of NADPH Cytochrome P450 Oxidoreductase. Frontiers in Pharmacology, 10, p. 1187. Frontiers 10.3389/fphar.2019.01187

[img]
Preview
Text
fphar-10-01187.pdf - Published Version
Available under License Creative Commons: Attribution (CC-BY).

Download (1MB) | Preview

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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Department of Gynaecology, Paediatrics and Endocrinology (DFKE) > Clinic of Paediatric Medicine
04 Faculty of Medicine > Department of Gynaecology, Paediatrics and Endocrinology (DFKE) > Clinic of Paediatric Medicine > Endocrinology/Metabolic Disorders
04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > Unit Childrens Hospital > Forschungsgruppe Endokrinologie / Diabetologie / Metabolik (Pädiatrie)

UniBE Contributor:

Parween, Shaheena; Udhane, Sameer Sopanrao and Pandey, Amit Vikram

Subjects:

600 Technology > 610 Medicine & health

ISSN:

1663-9812

Publisher:

Frontiers

Language:

English

Submitter:

Karen Lidzba

Date Deposited:

22 Jan 2020 12:09

Last Modified:

26 Jan 2020 02:50

Publisher DOI:

10.3389/fphar.2019.01187

PubMed ID:

31749697

Uncontrolled Keywords:

CYP2C19 CYP2C9 CYP3A4 CYP3A5 P450 oxidoreductase POR cytochrome P450 protein- protein interaction

BORIS DOI:

10.7892/boris.138673

URI:

https://boris.unibe.ch/id/eprint/138673

Actions (login required)

Edit item Edit item
Provide Feedback