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. 2024 May;121(5):1532-1542.
doi: 10.1002/bit.28662. Epub 2024 Jan 24.

Computer-directed rational design enhanced the thermostability of carbonyl reductase LsCR for the synthesis of ticagrelor precursor

Shen-Yuan Xu  1   2   3 , Rong-Liang Chu  1   2   3 , Hua-Tao Liu  1   2   3 , Chun-Yue Weng  1   2   3 , Ya-Jun Wang  1   2   3 , Yu-Guo Zheng  1   2   3
Affiliations

Computer-directed rational design enhanced the thermostability of carbonyl reductase LsCR for the synthesis of ticagrelor precursor

Shen-Yuan Xu et al. Biotechnol Bioeng. 2024 May.

Abstract

Carbonyl reductases are useful for producing optically active alcohols from their corresponding prochiral ketones. Herein, we applied a computer-assisted strategy to increase the thermostability of a previously constructed carbonyl reductase, LsCRM4 (N101D/A117G/F147L/E145A), which showed an outstanding activity in the synthesis of the ticagrelor precursor (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol. The stability changes introduced by mutations at the flexible sites were predicted using the computational tools FoldX, I-Mutant 3.0, and DeepDDG, which demonstrated that 12 virtually screened mutants could be thermally stable; 11 of these mutants exhibited increased thermostability. Then a superior mutant LsCRM4-V99L/D150F was screened out from the library that was constructed by iteratively combining the beneficial sites, which showed a 78% increase in activity and a 17.4°C increase in melting temperature compared to LsCRM4. Our computer-assisted design and combinatorial strategy dramatically increased the efficiency of thermostable enzyme production.

Keywords: carbonyl reductase; chiral alcohol; computer‐aided design; protein engineering; thermostability.

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References

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