()-selective transaminases show promise as catalysts for the asymmetric synthesis of chiral amines, which are building blocks of various small molecule drugs. However, their application is limited by poor substrate acceptance and low catalytic efficiency. Here, a potential ()-selective transaminase from (FsTA) was identified through a substrate truncating strategy, and used as starting point for enzyme engineering toward catalysis of 4-hydroxy-2-butanone, a substrate that poses challenges in catalysis. Molecular docking and dynamics simulations revealed Y90 as the key residue responsible for poor substrate binding. Starting from the variant (Y90F, mut1) with initial activity, FsTA was systematically modified to improve substrate-binding through active site reshaping and consensus sequence strategy, yielding three variants (H30R, V152K, and Y156F) with improved activity. A quadruple mutation variant H30R/Y90F/V152K/Y156F (mut4) was also found to show a 7.95-fold greater catalytic efficiency (/) than the initial variant mut1. Furthermore, mut4 also enhanced the thermostability of enzyme significantly, with the value increasing by 10 °C. This variant also exhibited significantly improved activity toward a series of ketones that are either not accepted or poorly accepted by the wild-type. This study provides a basis for the rational design of an active to creating variants that can accommodate novel substrates.

中文翻译：

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工程化 (R)-选择性转氨酶用于不对称合成 (R)-3-氨基丁醇

()-选择性转氨酶有望作为手性胺不对称合成的催化剂，手性胺是各种小分子药物的组成部分。然而，它们的应用受到底物接受性差和催化效率低的限制。在这里，通过底物截短策略鉴定了来自 (FsTA) 的潜在 ()-选择性转氨酶，并将其用作催化 4-羟基-2-丁酮（一种对催化提出挑战的底物）的酶工程的起点。分子对接和动力学模拟表明，Y90 是导致底物结合不良的关键残基。从具有初始活性的变体（Y90F、mut1）开始，通过活性位点重塑和共有序列策略对 FsTA 进行系统性修饰以改善底物结合，产生具有改进活性的三个变体（H30R、V152K 和 Y156F）。还发现四重突变变体 H30R/Y90F/V152K/Y156F (mut4) 的催化效率 (/) 比初始变体 mut1 高 7.95 倍。此外，mut4还显着增强了酶的热稳定性，增加了10℃。该变体还表现出对野生型不接受或接受程度较差的一系列酮的显着改善的活性。这项研究为合理设计活性物质以创建可适应新型底物的变体提供了基础。