Testosterone(TS) is a male sterol hormone that regulates human hormone metabolism and also an important intermediate for synthesizing other steroid drugs. TS is prepared by reducing the carbonyl group on C-17 of 4-androstene-3,17-done (4-AD) to the hydroxyl group. Human-derived 17β-hydroxysteroid dehydrogenase type3 (17β-HSD3) was selected here for the regioselective and stereoselective catalytic reaction from 4-AD to TS in Pichia pastoris GS115. To solve the problem of poor catalytic activity of 17β-HSD3, we dissected the structure of 17β-HSD3 and analyzed its catalytic mechanism with the aid of computer assistance for the first time, and a mutant enzyme with significantly improved enzymatic properties was obtained after rationally designing the amino acid residues in the active center. First, the three-dimensional (3D) conformation of 17β-HSD3 was constructed by homology modeling using 17β-HSD1 (1FDT) as the template. Importantly, molecular dynamics (MD) simulations revealed structural features of the enzyme-substrate complex, which demonstrated that appropriate mutations of G186R/Y195W might induce higher stability and stronger interaction, conferring 17β-HSD3 a stronger ability to reduce 4-AD. Thus, we obtained the preferred mutant Picha-G186R/Y195W with 2.84-fold improvement in TS. This study not only provides an efficient mild biocatalyst for green production of TS, but also proposes a sound basis for elaborating the catalytic mechanism of 17β-HSD3.

Rational design of 17β-hydroxysteroid dehydrogenase type3 for improving testosterone production with an engineered Pichia pastoris GS115

ACS Spring Meeting 2021

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