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Nonconserved hinge in Baeyer-Villiger monooxygenase affects catalytic activity and stereoselectivity / 生物工程学报
Chinese Journal of Biotechnology ; (12): 361-374, 2015.
Article in Chinese | WPRIM | ID: wpr-240637
ABSTRACT
Baeyer-Villiger monooxygenases (BVMOs) are important biocatalysts to synthesize a series of valuable esters and lactones. Based on protein sequence alignment and crystal structure analysis, a nonconserved hinge which linked NADPH domain and FAD domain was speculated to play an important role in substrate recognition and catalytic oxidation process. Cyclohexanone monooxygenase (CHMO) was selected as a model. Mutants obtained by homologous replacement of the whole hinge almost completely lost its original catalytic activity, demonstrating that the overall hinge structure was of great importance. Some significant sites were identified to greatly affect the catalytic activity and stereoselectivity by alanine scanning mutagenesis, accompanied by enzyme activity assessments and chiral kinetic resolutions. Altering K153 decreased the activity of the enzyme but enhanced the stereoselectivity. Changing L143 site reduced stereoselectivity but had little effect on enzyme activity. Mutation at L144 site dramatically weakened both activity and stereoselectivity. Subsequently, these corresponding sites in phenylacetone monooxygenase were also illustrated to follow a similar rule, revealing a universal importance of these sites in the BVMO family. These results expanded our understanding of the structure-activity relationship of these enzymes and provided more proofs for future directed evolution of BVMOs.
Subject(s)
Full text: Available Index: WPRIM (Western Pacific) Main subject: Oxidation-Reduction / Oxygenases / Protein Conformation / Structure-Activity Relationship / Substrate Specificity / Models, Molecular / Catalysis / Chemistry / Sequence Alignment / Amino Acid Sequence Language: Chinese Journal: Chinese Journal of Biotechnology Year: 2015 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Main subject: Oxidation-Reduction / Oxygenases / Protein Conformation / Structure-Activity Relationship / Substrate Specificity / Models, Molecular / Catalysis / Chemistry / Sequence Alignment / Amino Acid Sequence Language: Chinese Journal: Chinese Journal of Biotechnology Year: 2015 Type: Article