Loss of Protein Stability and Function Caused by P228L Variation in NADPH-Cytochrome P450 Reductase Linked to Lower Testosterone Levels.

Rojas Velazquez, Maria Natalia; Noebauer, Mathias; Pandey, Amit Vikram (2022). Loss of Protein Stability and Function Caused by P228L Variation in NADPH-Cytochrome P450 Reductase Linked to Lower Testosterone Levels. International journal of molecular sciences, 23(17) MDPI 10.3390/ijms231710141

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Cytochrome P450 oxidoreductase (POR) is the redox partner of steroid and drug-metabolising cytochromes P450 located in the endoplasmic reticulum. Mutations in POR cause a broad range of metabolic disorders. The POR variant rs17853284 (P228L), identified by genome sequencing, has been linked to lower testosterone levels and reduced P450 activities. We expressed the POR wild type and the P228L variant in bacteria, purified the proteins, and performed protein stability and catalytic functional studies. Variant P228L affected the stability of the protein as evidenced by lower unfolding temperatures and higher sensitivity to urea denaturation. A significant decline in the rate of electron transfer to cytochrome c and thiazolyl blue tetrazolium (MTT) was observed with POR P228L, while activities of CYP3A4 were reduced by 25% and activities of CYP3A5 and CYP2C9 were reduced by more than 40% compared with WT POR. The 17,20 lyase activity of CYP17A1, responsible for the production of the main androgen precursor dehydroepiandrosterone, was reduced to 27% of WT in the presence of the P228L variant of POR. Based on in silico and in vitro studies, we predict that the change of proline to leucine may change the rigidity of the protein, causing conformational changes in POR, leading to altered electron transfer to redox partners. A single amino acid change can affect protein stability and cause a severe reduction in POR activity. Molecular characterisation of individual POR mutations is crucial for a better understanding of the impact on different redox partners of POR.

Item Type:

Journal Article (Original Article)


04 Faculty of Medicine > Department of Gynaecology, Paediatrics and Endocrinology (DFKE) > Clinic of Paediatric Medicine > Endocrinology/Metabolic Disorders

Graduate School:

Graduate School for Cellular and Biomedical Sciences (GCB)

UniBE Contributor:

Rojas Velazquez, Maria Natalia, Pandey, Amit Vikram


600 Technology > 610 Medicine & health
500 Science > 540 Chemistry
500 Science > 570 Life sciences; biology






[4] Swiss National Science Foundation ; [UNSPECIFIED] Novartis Foundation for Medical Biological Research ; [73] Swiss Government Excellence Scholarship




Amit Vikram Pandey

Date Deposited:

13 Sep 2022 12:46

Last Modified:

06 Jan 2023 18:15

Publisher DOI:


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Uncontrolled Keywords:

CYP3A4 POR congenital adrenal hyperplasia cytochrome P450 drug metabolism metabolic disorders protein stability





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