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

Abstract

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.

Engineering Electron Transfer Pathway of Cytochrome P450s

“…Mutations of cytochrome P450 BM3, such as P450BM3 M7 mutants and P450BM3 M9 mutants, have improved electron transfer [ 77 ]. Velazquez et al [ 78 ] expressed POR (OMIM:*124015, HNGC:9208) wild type and P228L variant in bacteria, and based on computer and in vitro studies, predicted that the change of proline to leucine might change the rigidity of protein, change the conformation of POR, and lead to a decrease in the rate of electron transfer to cytochrome c and thiazole blue tetrazole (MTT) significantly. Yu et al [ 79 ] used a colorimetric high-throughput screening (HTS) system with DDA as the true substrate to conduct directed evolution and obtained a P450 mutant (R14R/D629G) with higher activity.…”

Exploring Novel Variants of the Cytochrome P450 Reductase Gene (POR) from the Genome Aggregation Database by Integrating Bioinformatic Tools and Functional Assays

“…The selection of control variants involved an exhaustive review of the existing literature to identify POR gene variants with well-documented experimental data [7,[10][11][12][13][14][15][16]31]. These control variants were specifically chosen due to their established pathogenic or benign nature, supported by robust experimental evidence.…”

“…Disruption was achieved through sonication, yielding a clear lysate free of cellular debris. Membranes containing the over-expressed POR WT or variants were stored at −80 • C [11,14,15].…”

“…These P450 enzymes include CP21A2, CYP17A1, and CYP19A1 responsible for steroid biosynthesis, as well as CYP51A1 and CYP26B1 contributing to bone formation [7][8][9]. Homozygous or compound heterozygous mutations in the human POR gene (MIM: *124015) give rise to a diverse spectrum of clinical manifestations known as POR deficiency (PORD; MIM: #613571) in an autosomal recessive inheritance [7,[10][11][12][13][14][15][16][17][18]. The severest forms of PORD encompass congenital adrenal hyperplasia (CAH) [18][19][20][21], disruptions in sexual and pubertal development (DSD), and skeletal anomalies resembling Antley-Bixler syndrome with genital anomalies and disordered steroidogenesis (ABS1; MIM: #201750) [22][23][24][25].…”

The possible mechanisms of ferroptosis in sepsis-associated acquired weakness

“…Free PUFAs synthesize lipids via synthases and integrate with phospholipids to form PUFA-PL to maintain cell membrane stability ( Welch et al, 2022 ). Physiologically, cytochrome P450 oxidoreductase (POR) is located in the endoplasmic reticulum ( Koppula et al, 2021 ; Rojas Velazquez et al, 2022 ). The electrons of nicotinamide dinucleotide phosphate hydrogen (NAD(P)H) are transferred to downstream CYP450 ( Yan et al, 2021 ) via POR; however, some of these electrons react with oxygen during transfer, which results in H 2 O 2 production.…”