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Exploiting Cofactor Versatility to Convert a FAD‐Dependent Baeyer–Villiger Monooxygenase into a Ketoreductase
Author(s) -
Xu Jian,
Peng Yongzhen,
Wang Zhiguo,
Hu Yujing,
Fan Jiajie,
Zheng He,
Lin Xianfu,
Wu Qi
Publication year - 2019
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.201907606
Subject(s) - chemistry , monooxygenase , stereochemistry , cofactor , stereoselectivity , flavin group , biocatalysis , active site , flavoprotein , cyclohexanone , moiety , docking (animal) , directed evolution , enzyme , combinatorial chemistry , catalysis , cytochrome p450 , mutant , biochemistry , reaction mechanism , gene , medicine , nursing
Cyclohexanone monooxygenases (CHMOs) show very high catalytic specificity for natural Baeyer–Villiger (BV) reactions and promiscuous reduction reactions have not been reported to date. Wild‐type CHMO from Acinetobacter sp. NCIMB 9871 was found to possess an innate, promiscuous ability to reduce an aromatic α‐keto ester, but with poor yield and stereoselectivity. Structure‐guided, site‐directed mutagenesis drastically improved the catalytic carbonyl‐reduction activity (yield up to 99 %) and stereoselectivity ( ee up to 99 %), thereby converting this CHMO into a ketoreductase, which can reduce a range of differently substituted aromatic α‐keto esters. The improved, promiscuous reduction activity of the mutant enzyme in comparison to the wild‐type enzyme results from a decrease in the distance between the carbonyl moiety of the substrate and the hydrogen atom on N5 of the reduced flavin adenine dinucleotide (FAD) cofactor, as confirmed using docking and molecular dynamics simulations.