Journal
ACS CATALYSIS
Volume 11, Issue 22, Pages 14274-14283Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acscatal.1c04324
Keywords
< em > reductive amination <; em >; < em > amine dehydrogenase <; em >; < em > protein engineering <; em >; < em > substrate scope <; em >; < em > chiral amine <; em >
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Funding
- National Natural Science Foundation of China [21878085, 31971380, 21472045, 21871085]
- National Key Research and Development Program of China [2019YFA09005000]
- Fundamental Research Funds for the Central Universities [22221818014]
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Engineered amine dehydrogenase GkAmDH has been evolved in the laboratory to broaden its substrate scope for reductive amination of an extensive set of ketones, particularly showing higher activity toward bulky ketones. Mutants M3 and M8 can synthesize bulky chiral amines with high conversion, high enantiomeric excess, and high turnover number, providing key chiral intermediates for drug synthesis with promising thermostability.
Amine dehydrogenase-catalyzed reductive amination of prochiral ketones with ammonia is a promising method for the synthesis of optically pure amines in the pharmaceutical and fine chemical industries. However, previously reported amine dehydrogenases show restricted catalytic capacity toward bulky ketones, which limits their widespread applications toward the production of chiral amines. Herein, we expanded the substrate scope of an engineered amine dehydrogenase GkAmDH from Geobacillus kaustophilus via laboratory evolution for the reductive amination of an extensive set of ketones. Several beneficial mutants were identified with a up to 2.2 U mg-1 activity toward bulky benzylacetone, 110-fold higher than that of M0. Using the engineered M3 and M8, structurally diverse bulky chiral amines could be synthesized with up to >99% conversion, >99% ee, and up to 18,900 TON. Among them, two key chiral intermediates used in the synthesis of the drugs medroxalol and dilevalol were produced on a gram scale in up to 85% yield and >99% ee. Additionally, the engineered enzymes M3 and M8 displayed considerable thermostability with a half-life of more than three days at 50 degrees C. These results demonstrate that these engineered amine dehydrogenases are promising biocatalysts for the synthesis of chiral amines. Molecular dynamics simulations provide insights into how mutations improve the amination activity toward bulky ketones and the thermostability. Superscript/Subscript Available
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