Mutations Increasing Cofactor Affinity, Improve Stability and Activity of a Baeyer-Villiger Monooxygenase

The typically low thermodynamic and kinetic stability of enzymes is a bottleneck for their application in industrial synthesis. Baeyer-Villiger monooxygenases, which oxidize ketones to lactones using aerial oxygen, among other activities, suffer particularly from these instabilities. Previous effort...

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Detalhes bibliográficos
Autores: Mansouri, Hamid R., Gracia Carmona, Oriol, Jodlbauer, Julia, Schweiger, Lorenz, Fink, Michael J., Breslmayr, Erik, Laurent, Christophe, Feroz, Saima, Goncalves, Leticia C. P., Rial, Daniela Veronica, Mihovilovic, Marko D., Bommarius, Andreas S., Ludwig, Roland, Oostenbrink, Chris, Rudroff, Florian
Tipo de documento: artigo
Estado:Versão publicada
Data de publicação:2022
País:Argentina
Recursos:Consejo Nacional de Investigaciones Científicas y Técnicas
Repositório:CONICET Digital (CONICET)
Idioma:inglês
OAI Identifier:oai:ri.conicet.gov.ar:11336/218216
Acesso em linha:http://hdl.handle.net/11336/218216
Access Level:Acceso aberto
Palavra-chave:CYCLOHEXANONE MONOOXYGENASE
ENZYME STABILIZATION
MUTAGENESIS
OXIDATION
PROTEIN ENGINEERING
STRUCTURE-GUIDED CONSENSUS APPROACH
https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
Descrição
Resumo:The typically low thermodynamic and kinetic stability of enzymes is a bottleneck for their application in industrial synthesis. Baeyer-Villiger monooxygenases, which oxidize ketones to lactones using aerial oxygen, among other activities, suffer particularly from these instabilities. Previous efforts in protein engineering have increased thermodynamic stability but at the price of decreased activity. Here, we solved this trade-off by introducing mutations in a cyclohexanone monooxygenase from Acinetobacter sp., guided by a combination of rational and structure-guided consensus approaches. We developed variants with improved activity (1.5- to 2.5-fold) and increased thermodynamic (+5 °C Tm) and kinetic stability (8-fold). Our analysis revealed a crucial position in the cofactor binding domain, responsible for an 11-fold increase in affinity to the flavin cofactor, and explained using MD simulations. This gain in affinity was compatible with other mutations. While our study focused on a particular model enzyme, previous studies indicate that these findings are plausibly applicable to other BVMOs, and possibly to other flavin-dependent monooxygenases. These new design principles can inform the development of industrially robust, flavin-dependent biocatalysts for various oxidations.

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