Extending the Substrate Scope of an ω-Amine Transaminase from Aspergillus terreus by Reconstructing and Engineering an Ancestral Enzyme

作者：Tingting Cai, Jie Chen, Linquan Wang, Fangfang Fan, Guo Chen, Shuai Qiu, Na Li, Lehe Mei, Jun Huang · 发表于：ACS Catalysis · 年份：2025 · DOI：10.1021/acscatal.5c01779 · 被引用次数：18 · 研究领域：Enzyme Structure and Function、Amino Acid Enzymes and Metabolism、Enzyme Catalysis and Immobilization

Amine transaminases (ATAs) are used for synthesizing chiral amines from prochiral ketones or aldehydes through asymmetric reductive amination. However, there is still an urgent need to develop and evolve more ATAs with good performance, such as high activity, high stability, and wide substrate scope, to adapt to industrial production. Herein, a strategy of A ncestral S equence Reconstruction- C rystal Structure Guided- P ocket Engineering (ASCP) was used to engineer R -selective ω-ATA from Aspergillus terreus ( At ATA) for enhancing the thermostability and catalytic performance toward non-natural substrates. Through the ancestral sequence reconstruction (ASR) strategy, an ancestral ω-ATA (Anc101) was acquired, which showed a 10.9 °C enhancement in half-inactivation temperature ( T 50 10 ) and 484-fold improvement in half-life ( t 1/2 ) at 45 °C compared with At ATA. To increase the activities of Anc101 toward non-natural substrates, the substrate binding pocket was modified based on the X-ray crystal structure of Anc101, which we solved at a resolution of 2.3 Å (PDB: 8ZM7 ). The best mutant Anc1016 (Anc101-H55T-E117S-R128M-V150A-L183F-L188F) showed a 133-fold improvement in catalytic activity toward 3-acetylbiphenyl as compared with Anc101. The conversions using Anc1016 toward all tested substrates were increased by 2–87% compared with Anc101. Mechanism analysis revealed that the “gate ring” covering the cavity entrance of Anc1016 was more flexible than that in Anc101, thereb...