The effective synthesis of N-benzyl cyclo-tertiary amines using imine reductase, key components in natural products and pharmaceutical synthesis, is a green approach. Traditional methods faced challenges with enzyme activity and selectivity. This study focused on enhancing Mesorhizobium imine reductase (MesIRED) for better N-benzyl cyclo-tertiary amine production. Through alanine scanning and consensus mutation, 12 single-site MesIRED mutants were identified from 23 candidates, showing improved conversion of N-benzylpyrrolidine and N-benzylpiperidine. Notably, mutants from I177, V212, I213, and A241 significantly boosted conversions. The best-performing mutant for N-benzylpyrrolidine, MesIREDV212A/I213V (M1), increased conversion from 23.7% to 74.3%. For N-benzylpiperidine, MesIREDV212A/I177A/A241I (M2) enhanced conversion from 22.8% to 66.8%. Tunnel analysis revealed M1 and M2 have more efficient tunnels for larger product movement compared to wild-type MesIRED. Using recombinant E. coli coexpressing MesIRED and glucose dehydrogenase (GDH), high conversions were achieved: 75.1% for N-benzylpyrrolidine (M1) and 88.8% for N-benzylpiperidine (M2). A preparative experiment resulted in 86.2% conversion and 60.2% yield for N-benzylpiperidine. This research offers an efficient method for engineering IRED, significantly improving conversion and selectivity for N-benzyl cyclo-tertiary amines, aiding drug synthesis and providing insights into rational design of other enzymes.

中文翻译：

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合理改造中索根瘤菌亚胺还原酶以改进 N-苄基环叔胺的合成

利用亚胺还原酶有效合成天然产物和药物合成的关键成分N-苄基环叔胺是一种绿色方法。传统方法面临酶活性和选择性的挑战。本研究的重点是增强中索根瘤菌亚胺还原酶 (MesIRED)，以更好地生产 N-苄基环叔胺。通过丙氨酸扫描和共有突变，从 23 个候选者中鉴定出 12 个单位点 MesIRED 突变体，显示 N-苄基吡咯烷和 N-苄基哌啶的转化率有所提高。值得注意的是，I177、V212、I213 和 A241 的突变体显着提高了转化率。N-苄基吡咯烷的性能最佳突变体 MesIREDV212A/I213V (M1) 将转化率从 23.7% 提高到 74.3%。对于 N-苄基哌啶，MesIREDV212A/I177A/A241I (M2) 将转化率从 22.8% 提高到 66.8%。隧道分析显示，与野生型 MesIRED 相比，M1 和 M2 具有更高效的隧道，可实现更大的产品移动。使用共表达 MesIRED 和葡萄糖脱氢酶 (GDH) 的重组大肠杆菌，实现了高转化率：N-苄基吡咯烷 (M1) 为 75.1%，N-苄基哌啶 (M2) 为 88.8%。制备实验的结果是 N-苄基哌啶的转化率为 86.2%，产率为 60.2%。这项研究提供了一种有效的 IRED 工程方法，显着提高 N-苄基环叔胺的转化率和选择性，有助于药物合成，并为其他酶的合理设计提供见解。