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
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. E...
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| Format: | Article |
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Frontiers Media S.A.
2019-10-01
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| Series: | Frontiers in Pharmacology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fphar.2019.01187/full |
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| author | Shaheena Parween Shaheena Parween Maria Natalia Rojas Velazquez Maria Natalia Rojas Velazquez Maria Natalia Rojas Velazquez Sameer S. Udhane Sameer S. Udhane Norio Kagawa Amit V. Pandey Amit V. Pandey |
| author_facet | Shaheena Parween Shaheena Parween Maria Natalia Rojas Velazquez Maria Natalia Rojas Velazquez Maria Natalia Rojas Velazquez Sameer S. Udhane Sameer S. Udhane Norio Kagawa Amit V. Pandey Amit V. Pandey |
| author_sort | Shaheena Parween |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-20T22:08:16Z |
| format | Article |
| id | doaj.art-216fd4229aa54bb8b72fdae71347e5c0 |
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| issn | 1663-9812 |
| language | English |
| last_indexed | 2024-12-20T22:08:16Z |
| publishDate | 2019-10-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Pharmacology |
| spelling | doaj.art-216fd4229aa54bb8b72fdae71347e5c02022-12-21T19:25:13ZengFrontiers Media S.A.Frontiers in Pharmacology1663-98122019-10-011010.3389/fphar.2019.01187471401Variability in Loss of Multiple Enzyme Activities Due to the Human Genetic Variation P284T Located in the Flexible Hinge Region of NADPH Cytochrome P450 OxidoreductaseShaheena Parween0Shaheena Parween1Maria Natalia Rojas Velazquez2Maria Natalia Rojas Velazquez3Maria Natalia Rojas Velazquez4Sameer S. Udhane5Sameer S. Udhane6Norio Kagawa7Amit V. Pandey8Amit V. Pandey9Pediatric Endocrinology, Diabetology, and Metabolism, Department of Pediatrics, University Children’s Hospital Bern, Bern, SwitzerlandDepartment of Biomedical Research, University of Bern, Bern, SwitzerlandPediatric Endocrinology, Diabetology, and Metabolism, Department of Pediatrics, University Children’s Hospital Bern, Bern, SwitzerlandDepartment of Biomedical Research, University of Bern, Bern, SwitzerlandInstituto de Investigaciones en Ciencias de la Salud, Universidad Nacional de Asunción, San Lorenzo, ParaguayPediatric Endocrinology, Diabetology, and Metabolism, Department of Pediatrics, University Children’s Hospital Bern, Bern, SwitzerlandDepartment of Biomedical Research, University of Bern, Bern, SwitzerlandSchool of Medicine, Nagoya University, Nagoya, JapanPediatric Endocrinology, Diabetology, and Metabolism, Department of Pediatrics, University Children’s Hospital Bern, Bern, SwitzerlandDepartment of Biomedical Research, University of Bern, Bern, SwitzerlandCytochromes 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.https://www.frontiersin.org/article/10.3389/fphar.2019.01187/fullPORP450 oxidoreductaseCYP2C9CYP2C19CYP3A4CYP3A5 |
| spellingShingle | Shaheena Parween Shaheena Parween Maria Natalia Rojas Velazquez Maria Natalia Rojas Velazquez Maria Natalia Rojas Velazquez Sameer S. Udhane Sameer S. Udhane Norio Kagawa Amit V. Pandey Amit V. Pandey 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 POR P450 oxidoreductase CYP2C9 CYP2C19 CYP3A4 CYP3A5 |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| topic | POR P450 oxidoreductase CYP2C9 CYP2C19 CYP3A4 CYP3A5 |
| url | https://www.frontiersin.org/article/10.3389/fphar.2019.01187/full |
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