ω-Transaminase (ω-TA) is an attractive biocatalyst for stereospecific preparation of amino acids and derivatives, but low catalytic efficiency and unfavorable substrate specificity hamper their industrial application. In this work, to obtain applicable (R)-ω-TA responsible for amination of α-keto acids substrates, the reactivities of eight previously synthesized ω-TAs toward pyruvate using (R)-α-methylbenzylamine ((R)-α-MBA) as amine donor were investigated, and Gibberella zeae TA (GzTA) with the highest (R)-TA activity and stereoselectivity was selected as starting scaffold for engineering. Site-directed mutagenesis around enzymatic active pocket and access tunnel identified three positive mutation sites, S214A, F113L, and V60A. Kinetic analysis synchronously with molecular docking revealed that these mutations afforded desirable alleviation of steric hindrance for pyruvate and α-MBA. Furthermore, the constructed single-, double-, and triple-mutant exhibited varying degrees of improved specificities toward bulkier α-keto acids. Using 2-oxo-2-phenylacetic acid (1d) as substrate, the conversion rate of triple-mutant F113L/V60A/S214A increased by 3.8-fold relative to that of wide-type GzTA. This study provided a practical engineering strategy for improving catalytic efficiency and substrate specificity of (R)-ω-TA. The obtained experience shed light on creating more industrial ω-TAs mutants that can accommodate structurally diverse substrates.

Graphical abstract

中文翻译：

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(R)-Omega-转氨酶的再设计及其在大侧链氨基酸合成中的应用

ω-转氨酶（ω-TA）是一种有吸引力的用于立体特异性制备氨基酸和衍生物的生物催化剂，但催化效率低和底物特异性不好阻碍了其工业应用。在这项工作中，为了获得适用于 α-酮酸底物胺化的 ( R )-ω-TA，使用 ( R )-α-甲基苄胺 (( R )-α- MBA) 作为胺供体进行了研究,玉米赤霉TA (GzTA) 具有最高 ( R)-TA 活性和立体选择性被选为工程的起始支架。围绕酶活性口袋和通路隧道的定点诱变确定了三个阳性突变位点，S214A、F113L 和 V60A。与分子对接同步的动力学分析表明，这些突变为丙酮酸和 α-MBA 提供了理想的空间位阻缓解。此外，构建的单、双和三突变体对更大的α-酮酸表现出不同程度的改进的特异性。以2-氧代-2-苯乙酸( 1d )为底物，三突变体F113L/V60A/S214A的转化率相对于宽型GzTA提高了3.8倍。本研究提供了一种实用的工程策略，可提高 (R )-ω-TA。所获得的经验为创造更多的工业 ω-TAs 突变体提供了启示，这些突变体可以适应结构多样化的底物。

图形概要