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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Detalles 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 recurso: artículo
Estado:Versión publicada
Fecha de publicación:2022
País:Argentina
Institución:Consejo Nacional de Investigaciones Científicas y Técnicas
Repositorio:CONICET Digital (CONICET)
Idioma:inglés
OAI Identifier:oai:ri.conicet.gov.ar:11336/218216
Acceso en línea:http://hdl.handle.net/11336/218216
Access Level:acceso abierto
Palabra clave: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
Descripción
Sumario: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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