Ancestor-originated engineering of medium-chain alcohol dehydrogenase enhances its catalytic compatibility by balancing activity, stability, and selectivity trade-offs

IF 3.9 2区化学 Q2 CHEMISTRY, PHYSICAL

Molecular Catalysis Pub Date : 2024-11-07 DOI:10.1016/j.mcat.2024.114657

Jun Wang , Lei Qin , Jie Gu , Lunjie Wu , Man Zou , Xin Su , Yan Xu , Yao Nie

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引用次数: 0

Abstract

The role of medium-chain alcohol dehydrogenases (MDR) in the preparation of chiral drug intermediates is indispensable; however, limited activity and stability towards unnatural substrates are crucial factors that restrict their application. Further, research on the ancestral MDRs remains unclear. Therefore, enhancing the activity, selectivity, and stability of MDRs is necessary. In this study, we resurrected ancestors of the MDR family using ancestral sequence reconstruction with descendant GcADH as a probe. Among these ancestors, ancestor 136 exhibited increased thermal stability (ΔT_m = 19.5 °C) and had a comparable conversion and selectivity as GcADH to the selected substrates. After in-silico screening using the Funclib tool, we obtained the mutant anc136-R2, which exhibited strengthened selectivity and activity towards N-Benzyl-3,3-difluoropiperidin-4-one (1a). Moreover, the mutations of anc136-R2 were simultaneously mapped to GcADH, resulting GcADH-R2. Based on the results of substrate spectrum characterisation, anc136 was more receptive to mutations. Specifically, for the substrates N-Boc-3-piperidone (4a) and N-boc-3-oxoazepane (7a), the conversion rates of anc136-R2 were 29 and 57 times higher, respectively, than those of GcADH-R2, without compromising selectivity. In conclusion, this work provides guidance for exploring the evolutionary trajectory of the MDR family and further illustrates the superior balanced “trade-off” of activity–selectivity–stability on ancestral enzymes.

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中链醇脱氢酶的祖先工程学通过平衡活性、稳定性和选择性之间的权衡，增强了其催化兼容性

中链醇脱氢酶（MDR）在手性药物中间体的制备中发挥着不可或缺的作用；然而，对非天然底物的有限活性和稳定性是限制其应用的关键因素。此外，有关祖先 MDR 的研究仍不清楚。因此，有必要提高 MDRs 的活性、选择性和稳定性。在本研究中，我们以后代 GcADH 为探针，利用祖先序列重建技术复活了 MDR 家族的祖先。在这些祖先中，祖先 136 表现出更高的热稳定性（ΔTm = 19.5 °C），对所选底物的转化率和选择性与 GcADH 相当。使用 Funclib 工具进行内筛选后，我们得到了突变体 anc136-R2，它对 N-苄基-3,3-二氟哌啶-4-酮 (1a) 的选择性和活性都有所提高。此外，anc136-R2 的突变也同时映射到了 GcADH 上，得到了 GcADH-R2。根据底物谱分析的结果，anc136 更容易接受突变。具体来说，对于底物 N-叔丁氧羰基-3-哌啶酮（4a）和 N-叔丁氧羰基-3-氧氮杂环庚烷（7a），anc136-R2 的转化率分别是 GcADH-R2 的 29 倍和 57 倍，而且不会影响选择性。总之，这项工作为探索 MDR 家族的进化轨迹提供了指导，并进一步说明了祖先酶在活性-选择性-稳定性之间优越的平衡 "权衡"。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Molecular Catalysis Chemical Engineering-Process Chemistry and Technology

CiteScore

6.90

自引率

10.90%

发文量

700

审稿时长

40 days

期刊介绍： Molecular Catalysis publishes full papers that are original, rigorous, and scholarly contributions examining the molecular and atomic aspects of catalytic activation and reaction mechanisms. The fields covered are: Heterogeneous catalysis including immobilized molecular catalysts Homogeneous catalysis including organocatalysis, organometallic catalysis and biocatalysis Photo- and electrochemistry Theoretical aspects of catalysis analyzed by computational methods