Enzyme-catalyzed stereodivergent synthesis to access all possible stereoisomers of organofluorine compounds bearing multiple stereogenic centers remains an important and challenging subject. By integrative data-driven mining and mechanism-guided engineering of ketoreductases, we identified a stereodivergent biocatalytic platform to produce four stereoisomeric fluoroalkyl amino acid esters bearing two vicinal stereocenters. Fast triple-parameter coevolution via a semirational CAST/ISM strategy provided the quadruple mutant M5 (A140K/L203T/G92A/V84I) of ketoreductase BgADH not only displayed high stereoselectivity toward the target stereoisomers (99:1 dr, 99% ee) but also observed with enhanced activity (k_cat/K_m, 6.3 folds) and improved thermostability (T₅₀¹⁵, 4 °C). Crystal structural analysis and molecular dynamics (MD) simulation studies unveil two residues (A140 and F148) of BgADH to be the key sites that are responsible for the control of the stereoselectivity. The L203T/G92A mutation enhanced activity by affecting the conformational distribution of the α-helix within the active-site region, and V84I improved thermal stability by strengthening the hydrogen bonding network with neighboring residues. The synthetic utility was further demonstrated by fluoroalkyl substrate scope expansion, gram-scale reactions (648 g L^–1 day^–1), and synthetic transformations to chiral fluorinated β-lactams that are the antibiotic carbapenem cores.

中文翻译：

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通过三参数工程酮还原酶酶促立体发散获得氟化 β-内酰胺药效团

酶催化立体发散合成以获得具有多个立体中心的有机氟化合物的所有可能的立体异构体仍然是一个重要且具有挑战性的课题。通过综合数据驱动的挖掘和机制引导的酮还原酶工程，我们确定了一个立体发散的生物催化平台，可以生产带有两个邻位立体中心的四种立体异构氟烷基氨基酸酯。通过半理性CAST / ISM策略的快速三参数协同进化提供了酮还原酶Bg ADH的四重突变体 M5 (A140K/L203T/G92A/V84I)不仅对目标立体异构体表现出高立体选择性 (99:1 dr, 99% ee)，而且还观察到活性增强（k _cat / K _m， 6.3 倍）和热稳定性改善（T ₅₀ ¹⁵， 4 °C）。晶体结构分析和分子动力学 (MD) 模拟研究揭示了Bg ADH 的两个残基（A140 和 F148）是负责控制立体选择性的关键位点。 L203T/G92A 突变通过影响活性位点区域内 α 螺旋的构象分布来增强活性，V84I 通过加强与邻近残基的氢键网络来提高热稳定性。通过氟烷基底物范围扩展、克级反应（648 g L ^–1 day ^–1）以及作为抗生素碳青霉烯核心的手性氟化 β-内酰胺的合成转化，进一步证明了合成效用。