Ketoreductases are tools for the synthesis of chiral alcohols in industry. However, the low activity of natural enzymes often restricts their use in industrial applications. On the basis of computational analysis and previous reports, two residues (F92 and F94) probably affecting the activity of ketoreductase CgKR1 were identified. By tuning these two residues, the CgKR1-F92C/F94W variant was obtained that exhibited higher activity toward all 28 structurally diverse substrates examined than the wild-type enzyme. Among them, 13 substrates have a specific activity over 50 U mg^–1 (54–775 U mg^–1). Using CgKR1-F92C/F94W as a catalyst, five substrates at high loading (>100 g^–1 L^–1) were reduced completely in gram-scale preparative reactions. This approach provides accesses to pharmaceutically relevant chiral alcohols with high enantioselectivity (up to 99.0% ee) and high space-time yield (up to 583 g^–1 L^–1 day^–1). Molecular dynamics simulations highlighted the crucial role of residues 92 and 94 in activity improvement. Our findings provide useful guidance for engineering other ketoreductases, especially those possessing a similar active pocket to that in CgKR1.

中文翻译：

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工程酮还原酶Cg KR1制备结构多样的手性醇

酮还原酶是工业上合成手性醇的工具。然而，天然酶的低活性经常限制了它们在工业应用中的使用。根据计算分析和以前的报告，确定了可能影响酮还原酶Cg KR1活性的两个残基（F92和F94）。通过调节这两个残基，获得了Cg KR1-F92C / F94W变体，该变体对所检查的所有28种结构多样的底物均显示出比野生型酶更高的活性。其中，13种底物的比活度超过50 U mg ^–1（54–775 U mg ^–1）。使用Cg KR1-F92C / F94W作为催化剂，可在高负载下（> 100 g ^–1 L^–1）在克级制备反应中被完全还原。这种方法提供了具有高对映选择性（高达99.0％ee）和高时空产率（高达583 g ^–1 L ^–1天^–1）的药学上相关的手性醇。分子动力学模拟突出了残基92和94在提高活性中的关键作用。我们的发现为工程化其他酮还原酶提供了有用的指导，尤其是那些与Cg KR1具有相似活性口袋的酮还原酶。