Semi-rational engineering of a thermostable aldo–keto reductase from Thermotoga maritima for synthesis of enantiopure ethyl-2-hydroxy-4-phenylbutyrate (EHPB)

• Published in: Scientific reports Vol. 7; no. 1; pp. 4007 - 11
• Main Authors: Wang, Zhiguo, Zhou, Shuo, Zhang, Shuangling, Zhang, Sa, Zhu, Fangmeng, Jin, Xiaolu, Chen, Zhenming, Xu, Xiaoling
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 21.06.2017; Nature Publishing Group; Nature Portfolio
• Subjects: 119/118; 631/114/2397; 631/45/603; 631/45/607/1168; 631/61/338/469; 82/16; 82/80; 82/83; Aldo-keto reductase; Aldo-Keto Reductases - chemistry; Aldo-Keto Reductases - genetics; Angiotensin; Biocatalysts; Catalysts; Crystal structure; Crystallography, X-Ray; Enantiomers; Enzyme Stability; Enzymes - chemistry; Enzymes - genetics; Hot Temperature; Humanities and Social Sciences; multidisciplinary; Mutagenesis; NADP; Phenylbutyrates - chemistry; Phenylbutyric acid; Protein Engineering; Saturation mutagenesis; Science; Science (multidisciplinary); Substrate Specificity; Thermotoga maritima - enzymology; Thermotoga maritima - genetics
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-017-03947-8

A novel aldo-keto reductase Tm1743 characterized from Thermotoga maritima was explored as an effective biocatalyst in chiral alcohol production. Natural Tm1743 catalyzes asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (EOPB) at high efficiency, but the production of, ethyl (S)-2-hydroxy-4-phenylbutyrate ((S)-EHPB), which is less desirable, is preferred with an enantiomeric excess ( ee ) value of 76.5%. Thus, altering the enantioselectivity of Tm1743 to obtain the more valuable product (R)-EHPB for angiotensin drug synthesis is highly desired. In this work, we determined the crystal structure of Tm1743 in complex with its cofactor NADP + at 2.0 Å resolution, and investigated the enantioselectivity of Tm1743 through semi-rational enzyme design. Molecular simulations based on the crystal structure obtained two binding models representing the pro - S and pro - R conformations of EOPB. Saturation mutagenesis studies revealed that Trp21 and Trp86 play important roles in determining the enantioselectivity of Tm1743. The best (R)- and (S)-EHPB preferring Tm1743 mutants, denoted as W21S/W86E and W21L/W118H, were identified; their ee values are 99.4% and 99.6% and the catalytic efficiencies are 0.81 and 0.12 mM −1 s −1 , respectively. Our work presents an efficient strategy to improve the enantioselectivity of a natural biocatalyst, which will serve as a guide for further exploration of new green catalysts for asymmetric reactions.