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首页> 外文期刊>Journal of Molecular Biology >Human 20alpha-hydroxysteroid dehydrogenase: crystallographic and site-directed mutagenesis studies lead to the identification of an alternative binding site for C21-steroids.
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Human 20alpha-hydroxysteroid dehydrogenase: crystallographic and site-directed mutagenesis studies lead to the identification of an alternative binding site for C21-steroids.

机译：人20α-羟基类固醇脱氢酶：晶体学和定点诱变研究导致鉴定C21-类固醇的替代结合位点。

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摘要

Human 20alpha-hydroxysteroid dehydrogenase (h20alpha-HSD; AKR1C1) catalyzes the transformation of progesterone (Prog) into 20alpha-hydroxy-progesterone (20alpha-OHProg). Although h20alpha-HSD shares 98% sequence identity with human type 3 3alpha-HSD (h3alpha-HSD3, AKR1C2), these two enzymes differ greatly in their activities. In order to explain these differences, we have solved the crystal structure of h20alpha-HSD in a ternary complex with NADP(+) and 20alpha-OHProg at 1.59A resolution. The steroid is stabilized by numerous hydrophobic interactions and a hydrogen bond between its O20 and the N(epsilon ) atom of His222. This new interaction prevents the formation of a hydrogen bond with the cofactor, as seen in h3alpha-HSD3 ternary complexes. By combining structural, direct mutagenesis and kinetic studies, we found that the H(222)I substitution decreases the K(m) value for the cofactor 95-fold. With these results, we hypothesize that the rotation of the lateral chain of His222 could be a mediating step between the transformation of Prog and the release of the cofactor. Moreover, crystal structure analysis and direct mutagenesis experiments lead us to identify a new residue involved in the binding of Prog. Indeed, the R(304)L substitution leads to a 65-fold decrease in the K(m) value for Prog reduction. We thus propose that Prog is maintained in a new steroid-binding site composed mainly of residues found in the carboxy-terminal region of the protein.
机译：人20α-羟基甾体脱氢酶（h20alpha-HSD; AKR1C1）催化将孕酮（Prog）转化为20α-羟基孕酮（20alpha-OHProg）。尽管h20alpha-HSD与人类3型3alpha-HSD（h3alpha-HSD3，AKR1C2）具有98％的序列同一性，但这两种酶的活性差异很大。为了解释这些差异，我们以1.59A的分辨率解决了带有NADP（+）和20alpha-OHProg的三元复合物中h20alpha-HSD的晶体结构。类固醇通过许多疏水相互作用和其O20与His222的N（ε）原子之间的氢键稳定。如h3alpha-HSD3三元复合物中所见，这种新的相互作用可防止与辅因子形成氢键。通过结合结构，直接诱变和动力学研究，我们发现H（222）I替代降低了辅助因子的K（m）值95倍。根据这些结果，我们假设His222侧链的旋转可能是Prog转化与辅因子释放之间的中介步骤。此外，晶体结构分析和直接诱变实验使我们确定了参与Prog结合的新残基。实际上，R（304）L取代导致Prog降低的K（m）值降低65倍。因此，我们建议将Prog维持在新的类固醇结合位点中，该位点主要由在蛋白质的羧基末端区域中发现的残基组成。

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