Pediatric Endocrinology Unit, Department of Pediatrics, University Children's Hospital Bern, 3010 Bern, Switzerland.
Translational Hormone Research, Department of Biomedical Research, University of Bern, 3010 Bern, Switzerland.
Int J Mol Sci. 2022 Sep 4;23(17):10141. doi: 10.3390/ijms231710141.
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.
细胞色素 P450 氧化还原酶（POR）是位于内质网中的类固醇和药物代谢细胞色素 P450 的氧化还原伴侣。POR 中的突变会导致广泛的代谢紊乱。通过全基因组测序鉴定的 POR 变体 rs17853284（P228L）与较低的睾丸激素水平和降低的 P450 活性有关。我们在细菌中表达了 POR 野生型和 P228L 变体，纯化了蛋白质，并进行了蛋白质稳定性和催化功能研究。变体 P228L 影响了蛋白质的稳定性，表现在较低的解折叠温度和对脲变性的更高敏感性。与 POR WT 相比，POR P228L 与细胞色素 c 和噻唑蓝（MTT）的电子转移速率显著下降，而 CYP3A4 的活性降低了 25%，CYP3A5 和 CYP2C9 的活性降低了 40%以上。CYP17A1 的 17,20 裂解酶活性负责产生主要雄激素前体脱氢表雄酮，在 POR P228L 变体存在的情况下，该活性降低到 WT 的 27%。基于计算机模拟和体外研究，我们预测脯氨酸到亮氨酸的变化可能会改变蛋白质的刚性，导致 POR 构象发生变化，从而导致电子向氧化还原伴侣的转移发生改变。单个氨基酸的变化会影响蛋白质的稳定性，并导致 POR 活性严重降低。对 POR 中单个突变的分子特征分析对于更好地理解其对不同氧化还原伴侣的影响至关重要。
