Ding Juanjuan, You Shengping, Zhang Jiaxing, Zhang Hongtao, Wang Hui, Zhang Wei, Qi Wei, Su Rongxin, He Zhimin
Chemical Engineering Research Center, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, PR China.
Chemical Engineering Research Center, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, PR China; Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin University, Tianjin 300072, PR China.
Bioresour Technol. 2021 Dec;341:125833. doi: 10.1016/j.biortech.2021.125833. Epub 2021 Aug 25.
Testosterone (TS) is a critical androgenic steroid that regulates human metabolism and maintains secondary sexual characteristics. The biotransformation from 4-androstene-3,17-done (4-AD) to TS is limited by the poor catalytic activity of 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3). Herein, we explored the structural characteristics and catalytic mechanism of 17β-HSD3 and adopted the rational design strategy to improve its catalytic activity. Molecular docking and molecular dynamics simulations revealed the substrate-binding pocket and the binding mode of 4-AD to 17β-HSD3. We located the pivotal residues and regulated their hydrophobicity and polarity. The obtained G186R/Y195W variant formed additional electrostatic interaction and hydrogen bond with 4-AD, increasing the binding affinity between the variant and 4-AD. Therefore, the G186R/Y195W variant produced 3.98 g/L of TS, which increased to 297%. The combination of structural and mechanism resolution drives the implementation of the rational design strategy, which provides guidance for bioproduction of TS catalyzed by 17β-HSD3.
睾酮（TS）是一种关键的雄激素类固醇，可调节人体新陈代谢并维持第二性征。从4-雄烯-3,17-二酮（4-AD）到TS的生物转化受到3型17β-羟基类固醇脱氢酶（17β-HSD3）催化活性不佳的限制。在此，我们探究了17β-HSD3的结构特征和催化机制，并采用合理设计策略来提高其催化活性。分子对接和分子动力学模拟揭示了底物结合口袋以及4-AD与17β-HSD3的结合模式。我们确定了关键残基并调节了它们的疏水性和极性。获得的G186R/Y195W变体与4-AD形成了额外的静电相互作用和氢键，增加了变体与4-AD之间的结合亲和力。因此，G186R/Y195W变体产生了3.98 g/L的TS，提高到了297%。结构解析和机制解析的结合推动了合理设计策略的实施，为17β-HSD3催化TS的生物生产提供了指导。
