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

作     者：Jiaren Cao Fangfang Fan Changjiang Lyu Sheng Hu Weirui Zhao Jiaqi Mei Shuai Qiu Lehe Mei Jun Huang 

作者机构：Key Laboratory of Chemical and Biological Processing Technology for Farm Products of Zhejiang ProvinceZhejiang Provincial Collaborative Innovation Center of Agricultural Biological Resources Biochemical ManufacturingSchool of Biological and Chemical EngineeringZhejiang University of Science and TechnologyHangzhou 310023China School of Biological and Chemical EngineeringNingbo Tech UniversityNingbo 315100China Hangzhou Huadong Medicine Group Co.Ltd.Hangzhou 310011China College of Chemical and Biological EngineeringZhejiang UniversityHangzhou 310027China Jinhua Advanced Research InstituteJinhua 321019China 

出 版 物：《Engineering》 (工程（英文）)

年 卷 期：2023年第11期

页      面：203-214页

核心收录：

学科分类：081704[工学-应用化学] 07[理学] 08[工学] 0817[工学-化学工程与技术] 070303[理学-有机化学] 0703[理学-化学] 

基　　金：National Natural Science Foundation of China(32071268 and 31971372) the Ningbo"Scientific and Technological Innovation 2025"Key Project(2020Z080)for financial support 

主　　题：Trade-off Co-evolution Amine transaminase (R)-(+)-1(1-naphthyl)ethylamine 

摘      要：Amine transaminases(ATAs)catalyze the asymmetric amination of prochiral ketones or aldehydes to their corresponding chiral ***,the trade-off between activity and stability in enzyme engineering represents a major obstacle to the practical application of *** this trade-off is important for developing robustly engineered enzymes and a universal approach for ***,we modified the binding pocket of co-ATA from Aspergillus terreus(AtATA)to identify the key amino acid residues controlling the activity and stability of AtATA toward *** discovered a structural switch comprising four key amino acid sites(R128,V149,L182,and L187),as well as thebestmutant(AtATAD224K/V149A/L182 F/L187F;termed M4).Compared to the parent enzyme AtATAD224K(AtATAPa),M4 increased the catalytic efficiency(k_(cat)/K_(m)^(1-acetonaphthone),where kcatis the constant of catalytic activities and is 10.1 min^(-1),K_(m)^(1-acetonaphthoneis) 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-1and by 1.6-fold to 46.9 min,***,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(R99.5%)within 10 h.M4 also displayed significantly enhanced activity toward various 1-acetonaphthone *** related structural properties derived by analyzing structure and sequence information of robust ATAs illustrated their enhanced activity and *** of intramolecular interactions and expansion of the angle between the substratebinding pocket and the pyridoxal 5’-phosphate(PLP)-binding pocket contributed to synchronous enhancement of ATA thermostability and ***,this pocket engineering strategy successfully transferred enhanced activity and thermostability to three other ATAs,which exhibited 8%-22%sequence simila