Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, School of Life Sciences, Jilin University, Changchun, 130012, China.
Microb Cell Fact. 2023 Oct 15;22(1):213. doi: 10.1186/s12934-023-02225-9.
Enantio-pure α-hydroxy amides are valuable intermediates for the synthesis of chiral pharmaceuticals. The asymmetric reduction of α-keto amides to generate chiral α-hydroxy amides is a difficult and challenging task in biocatalysis. In this study, iolS, an aldo-keto reductase from Bacillus subtilis 168 was exhibited as a potential biocatalyst, which could catalyze the reduction of diaryl α-keto amide such as 2-oxo-N, 2-diphenyl-acetamide (ONDPA) with moderate S-selectivity (76.1%, ee) and 60.5% conversion. Through semi-rational engineering, two stereocomplementary variants (I57F/F126L and N21A/F126A) were obtained with ee value of 97.6% (S) and 99.9% (R) toward ONDPA (1a), respectively, delivering chiral α-hydroxy amide with > 98% conversions. Moreover, the excellent S- and R-preference variants displayed improved stereoselectivities toward the other α-keto amide compounds. Molecular dynamic and docking analysis revealed that the two key residues at 21 and 126 were identified as the "switch", which specifically controlled the stereopreference of iolS by regulating the shape of substrate binding pocket as well as the substrate orientation. Our results offer an effective strategy to obtain α-hydroxy amides with high optical purity and provide structural insights into altering the stereoselectivity of AKRs.
对映纯 α-羟基酰胺是手性药物合成的有价值的中间体。生物催化中，不对称还原 α-酮酰胺生成手性 α-羟基酰胺是一项困难且具有挑战性的任务。在这项研究中，枯草芽孢杆菌 168 的醛酮还原酶 iolS 被展示为一种潜在的生物催化剂，可催化二芳基 α-酮酰胺（如 2-氧代-N，2-二苯基-乙酰胺（ONDPA））的还原，具有中等的 S 选择性（76.1%，ee）和 60.5%的转化率。通过半理性工程，获得了两个立体互补变体（I57F/F126L 和 N21A/F126A），它们对 ONDPA（1a）的 ee 值分别为 97.6%（S）和 99.9%（R），可提供手性 α-羟基酰胺，转化率超过 98%。此外，这两种具有优异 S 和 R 偏好的变体对其他 α-酮酰胺化合物表现出了提高的立体选择性。分子动力学和对接分析表明，21 位和 126 位的两个关键残基被鉴定为“开关”，通过调节底物结合口袋的形状和底物取向，特异性控制 iolS 的立体选择性。我们的研究结果为获得高光学纯度的 α-羟基酰胺提供了一种有效的策略，并为改变 AKRs 的立体选择性提供了结构见解。
