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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 Ancestral Sequence Reconstruction-Crystal Structure Guided-Pocket Engineering (ASCP) was used to engineer R-selective ω-ATA from Aspergillus terreus (AtATA) 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 (T5010) and 484-fold improvement in half-life (t1/2) at 45 °C compared with AtATA. 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, thereby increasing access of the substrates to the binding pocket. In addition, the total volume of the large and small substrate binding pockets increased. Both of these alterations contribute to the enhanced activities toward non-natural substrates of Anc1016.
Cai et al. (Mon,) studied this question.