Vibegron functions as a potent and selective β3‐adrenergic receptor agonist, with its chiral precursor (2S,3R)‐aminohydroxy ester (1b) being crucial to its synthesis. In this study, loop engineering was applied to the carbonyl reductase (EaSDR6) from Exiguobacterium algae to achieve an asymmetric reduction of the (rac)‐aminoketone ester 1a. The variant M5 (A138L/A190V/S193A/Y201F/N204A) was obtained and demonstrated an 868‐fold increase in catalytic efficiency (kcat/Km = 260.3 s−1 mM−1) and a desirable stereoselectivity (>99% enantiomeric excess, e.e.; >99% diastereomeric excess, d.e.) for the target product 1b in contrast to the wild‐type EaSDR6 (WT). Structural alignment with WT indicated that loops 137–154 and 182–210 potentially play vital roles in facilitating catalysis and substrate binding. Moreover, molecular dynamics (MD) simulations of WT‐1a and M5‐1a complex illustrated that M5‐1a exhibits a more effective nucleophilic attack distance and more readily adopts a pre‐reaction state. The interaction analysis unveiled that M5 enhanced hydrophobic interactions with substrate 1a on cavities A and B while diminishing unfavorable hydrophilic interactions on cavity C. Computational analysis of binding free energies indicated that M5 displayed heightened affinity towards substrate 1a compared to the WT, aligning with its decreased Km value. Under organic‐aqueous biphasic conditions, the M5 mutant showed >99% conversion within 12 h with 300 g/L substrate 1a (highest substrate loading as reported). This study enhanced the catalytic performance of carbonyl reductase through functional loops engineering and established a robust framework for the large‐scale biosynthesis of the vibegron intermediate.

中文翻译：

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基于功能环工程的提高羰基还原酶的催化性能


Vibegron 是一种有效的选择性 β3-肾上腺素能受体激动剂，其手性前体 （2S，3R）-氨基羟基酯 （1b） 对其合成至关重要。在这项研究中，将循环工程应用于 Exiguobacterium 藻类的羰基还原酶 （EaSDR6），以实现 （rac）-氨基酮酯 1a 的不对称还原。获得变体 M5 （A138L/A190V/S193A/Y201F/N204A） 并证明与野生型 EaSDR6 （WT） 相比，目标产物 1b 的催化效率提高了 868 倍 （kcat/Km = 260.3 s-1 mM-1） 和理想的立体选择性（>99% 对映体过量，例如;>99% 非对映体过量，d.e.）。与 WT 的结构比对表明，环 137-154 和 182-210 可能在促进催化和底物结合方面发挥重要作用。此外，WT-1a 和 M5-1a 复合物的分子动力学 （MD） 模拟表明，M5-1a 表现出更有效的亲核攻击距离，并且更容易采用预反应状态。相互作用分析显示，M5 增强了空腔 A 和 B 上与底物 1a 的疏水相互作用，同时减少了空腔 C 上不利的亲水相互作用。结合自由能的计算分析表明，与 WT 相比，M5 对底物 1a 的亲和力更高，与其降低的 Km 值一致。在有机-水性双相条件下，M5 突变体在 300 g/L 底物 1a 下 12 小时内显示 >99% 转化（据报道，底物负载量最高）。本研究通过功能环工程增强了羰基还原酶的催化性能，并为 vibegron 中间体的大规模生物合成建立了稳健的框架。