氨基转移酶是一种能够催化前手性酮或醛不对称胺化生成相应的手性胺的生物催化剂。然而，活性和稳定性之间的“trade-off”效应限制了其在工业生产中的应用。因此，开发克服这种相互抵消的“trade-off”效应的普适方法以及获得高效催化的氨基转移酶具有重要意义。对此，我们针对土曲霉属氨基转移酶（AtATA）的底物结合口袋进行共进化改造，以确定控制AtATA对1-乙酰基萘活性和稳定性的关键氨基酸。通过多轮实验筛选，我们发现了一个含四个关键氨基酸位点（R128，V149，L182和L187）的“结构开关”， “最佳”突变体（AtATA_D224K/V149A/L182F/L187F; 命名为M4）。与亲本酶AtATA_D224K（AtATAPa）相比，M4的催化效率（k_cat/K_m^{1-乙酰基萘}，其中k_cat是催化活性常数，为10.1 min^-1，K_m^{1-乙酰基萘}是米氏常数，为1.7 mmol·L^-1）和半衰期（t_1/2）分别增加59倍和1.6倍。此外，在50 mL反应体系中，M4催化20 mmol·L^-1 1-乙酰基萘，反应10 h后可获得产率为78%的目标产物(R)-(+)-1-(1-萘基)乙胺（(R)-NEA），对映体纯度达到R > 99.5%。同时，M4还显示出对各种1-乙酰萘酮类似物显著增强的催化活性。通过结构和序列信息分析，进一步发现分子内相互作用的加强以及底物结合口袋和5’-磷酸吡哆醛（PLP）结合口袋之间的角度扩大是AtATA热稳定性和活性同步增强的重要原因。最后，口袋工程策略也将增强的活性和热稳定性成功地转移到其他三个序列相似性介于8%~22%之间的氨基转移酶中。这项研究对于克服ATA活性和热稳定性之间的权衡具有重要意义。

Amine transaminases (ATAs) catalyze the asymmetric amination of prochiral ketones or aldehydes to their corresponding chiral amines. However, the trade-off between activity and stability in enzyme engineering represents a major obstacle to the practical application of ATAs. Overcoming this trade-off is important for developing robustly engineered enzymes and a universal approach for ATAs. Herein, we modified the binding pocket of ω-ATA from Aspergillus terreus (AtATA) to identify the key amino acid residues controlling the activity and stability of AtATA toward 1-acetonaphthone. We discovered a structural switch comprising four key amino acid sites (R128, V149, L182, and L187), as well as the “best” mutant (AtATA_D224K/V149A/L182F/L187F; termed M4). Compared to the parent enzyme AtATA_D224K (AtATA-Pa), M4 increased the catalytic efficiency (k_cat/K_m^{1-acetonaphthone}, where k_cat is the constant of catalytic activities and is 10.1 min⁻¹, K_m^{1-acetonaphthone} is Michaelis-Menten constant and is 1.7 mmol·L^-1) and half-life (t_1/2) by 59-fold to 5.9 L·min⁻¹·mmol⁻¹ and by 1.6-fold to 46.9 min, respectively. Moreover, using M4 as the biocatalyst, we converted a 20 mmol·L^-1 aliquot of 1-acetonaphthone in a 50 mL scaled-up system to the desired product, (R)-(+)-1(1-naphthyl)ethylamine ((R)-NEA), with 78% yield and high enantiomeric purity (R > 99.5%) within 10 h. M4 also displayed significantly enhanced activity toward various 1-acetonaphthone analogs. The related structural properties derived by analyzing structure and sequence information of robust ATAs illustrated their enhanced activity and thermostability. Strengthening of intramolecular interactions and expansion of the angle between the substrate-binding pocket and the pyridoxal 5′-phosphate (PLP)-binding pocket contributed to synchronous enhancement of ATA thermostability and activity. Moreover, this pocket engineering strategy successfully transferred enhanced activity and thermostability to three other ATAs, which exhibited 8%-22% sequence similarity with AtATA. This research has important implications for overcoming the trade-off between ATA activity and thermostability.