酮甾体
脱氢
基质(水族馆)
脱氢酶
酶
化学
饱和突变
活动站点
氢化物
位阻效应
生物催化
试剂
产量(工程)
催化作用
立体化学
组合化学
有机化学
生物化学
反应机理
氢
异构酶
材料科学
海洋学
突变体
冶金
基因
地质学
作者
Yu Wang,Rui Zhang,Xi Chen,Zhonghua Yang,Jinhui Feng,Qiaqing Wu,Dunming Zhu
出处
期刊:ACS Sustainable Chemistry & Engineering
[American Chemical Society]
日期:2024-08-14
标识
DOI:10.1021/acssuschemeng.4c03609
摘要
Enzymatic Δ1-dehydrogenation mediated by 3-ketosteroid-Δ1-dehydrogenases (Δ1-KstDs, EC 1.3.99.4) offers an attractive method to access pharmaceutically important Δ1-3-ketosteroids by avoiding the multistep chemical reactions and use of toxic reagents. However, Δ1-KstDs have low or no activity toward C6-substituted and other bulky 3-ketosteroids, which limits their applications in the synthesis of the corresponding dehydrogenated products that are widely used for the treatment of different diseases. Herein, structure-based site-directed saturation mutagenesis of KstD from Propionibacterium sp. (PrKstD) was performed to tune its substrate specificity. Amino acid residues potentially responsible for substrate recognition were site-directedly mutated, and the results showed that the residues H135, A356, and S422 played important roles in fine-tuning the substrate specificity. Especially, the release of the steric effect of H135 provided extra space for accommodating the substrate with C6 methyl for a higher efficiency of transferring hydride at the active site. A double variant of PrKstD (H135T/A356N) exhibited 16.7-fold increased catalytic efficiency compared to that of the wild-type enzyme toward 6α-methyl-11β,17α-dihydroxy-4-pregnene-3,20-dione (1g). Molecular dynamics simulations provide some insights into the roles of the key mutations in the enhanced activity. Furthermore, Δ1-dehydrogenation of 6α-methyl-11β,17α-dihydroxy-4-pregnene-3,20-dione (1g) was scaled from gram to kilogram scale with high substrate loading (60.0 g·L–1) at a space–time yield of 4.08 g·L–1·h–1, much higher than the previously reported results. This work offers not only an effective method for the Δ1-dehydrogenation of C6-substituted 3-ketosteroids to furnish the corresponding bulky Δ1-3-ketosteroids but also guidance for tuning the substrate profile of 3-ketosteroid-Δ1-dehydrogenases to access other pharmaceutically relevant Δ1-3-ketosteroids in a green way.
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