Structure-based reshaping of a new ketoreductase from Sphingobacterium siyangense SY1 toward α-haloacetophenones.

Autor: Che C; School of Life Sciences and Biopharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe, Shenyang 110016, People's Republic of China., Zhang W; School of Life Sciences and Biopharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe, Shenyang 110016, People's Republic of China., Xu X; School of Life Sciences and Biopharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe, Shenyang 110016, People's Republic of China., Zheng Z; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin 300308, People's Republic of China., Wei H; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin 300308, People's Republic of China., Qin B; Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe, Shenyang 110016, People's Republic of China., Jia X; School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe, Shenyang 110016, People's Republic of China. Electronic address: jiaxian206@163.com., Liu W; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin 300308, People's Republic of China. Electronic address: liu_wd@tib.cas.cn., You S; School of Life Sciences and Biopharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe, Shenyang 110016, People's Republic of China. Electronic address: yousong206@aliyun.com.
Jazyk: angličtina
Zdroj: International journal of biological macromolecules [Int J Biol Macromol] 2024 Oct; Vol. 277 (Pt 1), pp. 134157. Date of Electronic Publication: 2024 Jul 24.
DOI: 10.1016/j.ijbiomac.2024.134157
Abstrakt: Ketoreductases play an indispensable role in the asymmetric synthesis of chiral drug intermediates, and an in-depth understanding of their substrate selectivity can improve the efficiency of enzyme engineering. In this endeavor, a new short-chain dehydrogenase/reductase (SDR) SsSDR1 identified from Sphingobacterium siyangense SY1 by gene mining method was successfully cloned and functionally expressed in Escherichia coli. Its activity against halogenated acetophenones has been tested and the results illustrated that SsSDR1-WT exhibits high activity for 3,5-bis(trifluoromethyl)acetophenone (1f), an important precursor in the synthesis of aprepitant. In addition, SsSDR1-WT showed obvious substrate preference for acetophenones without α-halogen substitution compared to their α-halogen analogs. To explore the structural basis of substrate selectivity, the X-ray crystal structures of SsSDR1-WT in its apo form and the complex structure with NAD were resolved. Taking 2-chloro-1-(3, 4-difluorophenyl) ethanone (1i) as the representative α-haloacetophenone, the key sites affecting substrate selectivity of SsSDR1-WT were identified and through the rational remodeling of the cavities C1 and C2 of SsSDR1, an excellent mutant I144A/S153L with significantly improved activity against α-halogenated acetophenones was obtained. The asymmetric catalysis of 1f and 1i was performed at the scale of 50 mL, and the space-time yields (STY) of the two were 1200 and 6000 g/L∙d, respectively. This study not only provides valuable biocatalysts for halogenated acetophenones, but also yields insights into the relationship between the substrate-binding pocket and substrate selectivity.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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