Pocket Modification of ω-Amine Transaminase AtATA for Overcoming the Trade-Off Between Activity and Stability Toward 1-Acetonaphthone
Jiaren Cao,
Fangfang Fan,
Changjiang Lyu,
Sheng Hu,
Weirui Zhao,
Jiaqi Mei,
Shuai Qiu,
Lehe Mei,
Jun Huang
Affiliations
Jiaren Cao
Key Laboratory of Chemical and Biological Processing Technology for Farm Products of Zhejiang Province, Zhejiang Provincial Collaborative Innovation Center of Agricultural Biological Resources Biochemical Manufacturing, School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, China
Fangfang Fan
Key Laboratory of Chemical and Biological Processing Technology for Farm Products of Zhejiang Province, Zhejiang Provincial Collaborative Innovation Center of Agricultural Biological Resources Biochemical Manufacturing, School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, China
Changjiang Lyu
Key Laboratory of Chemical and Biological Processing Technology for Farm Products of Zhejiang Province, Zhejiang Provincial Collaborative Innovation Center of Agricultural Biological Resources Biochemical Manufacturing, School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, China
Sheng Hu
School of Biological and Chemical Engineering, Ningbo Tech University, Ningbo 315100, China
Weirui Zhao
School of Biological and Chemical Engineering, Ningbo Tech University, Ningbo 315100, China
Jiaqi Mei
Hangzhou Huadong Medicine Group Co. Ltd., Hangzhou 310011, China
Shuai Qiu
Key Laboratory of Chemical and Biological Processing Technology for Farm Products of Zhejiang Province, Zhejiang Provincial Collaborative Innovation Center of Agricultural Biological Resources Biochemical Manufacturing, School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, China; Corresponding authors.
Lehe Mei
School of Biological and Chemical Engineering, Ningbo Tech University, Ningbo 315100, China; College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China; Jinhua Advanced Research Institute, Jinhua 321019, China; Corresponding authors.
Jun Huang
Key Laboratory of Chemical and Biological Processing Technology for Farm Products of Zhejiang Province, Zhejiang Provincial Collaborative Innovation Center of Agricultural Biological Resources Biochemical Manufacturing, School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, China; Corresponding authors.
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.
