Pocket Modification of ω-Amine Transaminase AtATA for Overcoming the Trade-Off Between Activity and Stability Toward 1-Acetonaphthone

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 (kcat/Km1-acetonaphthone, where kcat is the constant of catalytic activities and is 10.1 min−1, Km1-acetonaphthone is Michaelis-Menten constant and is 1.7 mmol·L–1) and half-life (t1/2) 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.