Laboratory of Molecular Endocrinology and Oncology, Centre Hospitalier Universitaire (CHU) de Quebec Research Center (CHUL) and Laval University, Québec City, Québec G1V4G2, Canada; Key Laboratory for Space Bioscience & Biotechnology, Institute of Special Environmental Biophysics, School of Life Sciences, Northwestern Polytechnical University, Xi'an 710072, PR China.
Laboratory of Molecular Endocrinology and Oncology, Centre Hospitalier Universitaire (CHU) de Quebec Research Center (CHUL) and Laval University, Québec City, Québec G1V4G2, Canada.
J Steroid Biochem Mol Biol. 2014 May;141:135-43. doi: 10.1016/j.jsbmb.2014.01.003. Epub 2014 Jan 13.
Human 3-alpha hydroxysteroid dehydrogenase type 3 (3α-HSD3) has an essential role in the inactivation of 5α-dihydrotestosterone (DHT). Notably, human 3α-HSD3 shares 97.8% sequence identity with human 20-alpha hydroxysteroid dehydrogenase (20α-HSD) and there is only one amino acid difference (residue 54) that is located in their steroid binding pockets. However, 20α-HSD displays a distinctive ability in transforming progesterone to 20α-hydroxy-progesterone (20α-OHProg). In this study, to understand the role of residue 54 in the steroid binding and discrimination, the V54L mutation in human 3α-HSD3 has been created. We have solved two crystal structures of the 3α-HSD3·NADP(+)·Progesterone complex and the 3α-HSD3 V54L·NADP(+)·progesterone complex. Interestingly, progesterone adopts two different binding modes to form complexes within the wild type enzyme, with one binding mode similar to the orientation of a bile acid (ursodeoxycholate) in the reported ternary complex of human 3α-HSD3·NADP(+)·ursodeoxycholate and the other binding mode resembling the orientation of 20α-OHProg in the ternary complex of human 20α-HSD·NADP(+)·20α-OHProg. However, the V54L mutation directly restricts the steroid binding modes to a unique one, which resembles the orientation of 20α-OHProg within human 20α-HSD. Furthermore, the kinetic study has been carried out. The results show that the V54L mutation significantly decreases the 3α-HSD activity for the reduction of DHT, while this mutation enhances the 20α-HSD activity to convert progesterone.
人 3-α 羟甾脱氢酶 3 型（3α-HSD3）在 5α-二氢睾酮（DHT）失活中具有重要作用。值得注意的是，人 3α-HSD3 与人 20-α 羟甾脱氢酶（20α-HSD）具有 97.8%的序列同一性，并且只有一个氨基酸差异（残基 54）位于它们的甾体结合口袋中。然而，20α-HSD 具有将孕酮转化为 20α-羟孕酮（20α-OHProg）的独特能力。在这项研究中，为了了解残基 54 在甾体结合和识别中的作用，创建了人 3α-HSD3 中的 V54L 突变。我们已经解决了人 3α-HSD3·NADP(+)·孕酮复合物和 3α-HSD3 V54L·NADP(+)·孕酮复合物的两个晶体结构。有趣的是，孕酮在野生型酶中采用两种不同的结合模式形成复合物，一种结合模式类似于报告的人 3α-HSD3·NADP(+)·熊去氧胆酸三元复合物中胆汁酸（熊去氧胆酸）的取向，另一种结合模式类似于人 20α-HSD·NADP(+)·20α-OHProg 三元复合物中 20α-OHProg 的取向。然而，V54L 突变直接将甾体结合模式限制为一种独特的模式，类似于人 20α-HSD 中 20α-OHProg 的取向。此外，还进行了动力学研究。结果表明，V54L 突变显著降低了 3α-HSD 对 DHT 的还原活性，而该突变增强了 20α-HSD 活性以转化孕酮。
