Enhancing the Activity of an Alcohol Dehydrogenase by Using “Aromatic Residue Scanning” at Potential Plasticity Sites

• Published in: Chemistry : a European journal Vol. 29; no. 25; pp. e202203530 - n/a
• Main Authors: Ye, Wen‐Jie, Xie, Jing‐Wen, Liu, Yan, Wang, Yi‐Lin, Zhang, Yu‐Xin, Yang, Xiao‐Ying, Yang, Lin, Wang, Hua‐Lei, Wei, Dong‐Zhi
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 02-05-2023
• Subjects: (S)-2-chloro-1-(3,4-difluorophenyl)ethanol; Alcohol dehydrogenase; Alcohol Dehydrogenase - genetics; Alcohol Dehydrogenase - metabolism; aromatic residue scanning; Binding Sites; Chemistry; Dehydrogenase; Drug tolerance; Ethanol; Mutagenesis; Phenylalanine; Plastic properties; Plasticity; plasticity sites; protein engineering; Residues; Scanning; Steric hindrance; Substrates; Tryptophan; Tyrosine; (S)-2-chloro-1-(3,4-difluorophenyl)ethanol; protein engineering; alcohol dehydrogenase; aromatic residue scanning; plasticity sites
• ISSN: 0947-6539; 1521-3765
• DOI: 10.1002/chem.202203530

An alcohol dehydrogenase LkADH was successfully engineered to exhibit improved activity and substrate tolerance for the production of (S)‐2‐chloro‐1‐(3,4‐difluorophenyl)ethanol, an important precursor of ticagrelor. Five potential hotspots were identified for enzyme mutagenesis by using natural residue abundance as an indicator to evaluate their potential plasticity. A semi‐rational strategy named “aromatic residue scanning” was applied to randomly mutate these five sites simultaneously by using tyrosine, tryptophan, and phenylalanine as “exploratory residues” to introduce steric hindrance or potential π‐π interactions. The best variant Lk‐S96Y/L199W identified with 17.2‐fold improvement in catalytic efficiency could completely reduce up to 600 g/L (3.1 M) 2‐chloro‐1‐(3,4‐difluorophenyl)ethenone in 12 h with >99.5 % ee, giving the highest space‐time yield ever reported. This study, therefore, offers a strategy for mutating alcohol dehydrogenase to reduce aromatic substrates and provides an efficient variant for the efficient synthesis of (S)‐2‐chloro‐1‐(3,4‐difluorophenyl)ethanol.
A strategy, named “aromatic residue scanning”, was developed for enzyme engineering, and an alcohol dehydrogenase was engineered to exhibit higher activity and substrate tolerance based on the implementation of the strategy to randomly mutate five identified potential plasticity sites. By using the best variant, an efficient bioprocess for the synthesis of (S)‐2‐chloro‐1‐(3,4‐difluorophenyl)ethanol was established.