Mutations increasing cofactor affinity, improve stability and activity of a Baeyer Villiger monooxygenase

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dc.citation.titleACS Catalysis
dc.citation.volume12
dc.contributor.orcidhttps://orcid.org/0000-0001-6560-9106
dc.contributor.orcidhttps://orcid.org/0000-0002-9944-1396
dc.contributor.orcidhttps://orcid.org/0000-0002-9112-6981
dc.contributor.orcidhttps://orcid.org/0000-0003-4658-1675
dc.contributor.orcidhttps://orcid.org/0000-0002-5058-5874
dc.contributor.orcidhttps://orcid.org/0000-0002-4232-2556
dc.contributor.orcidhttps://orcid.org/0000-0002-6680-8200
dc.creatorMansouri, Hamid R.
dc.creatorGracia Carmona, Oriol
dc.creatorJodlbauer, Julia
dc.creatorSchweiger, Lorenz
dc.creatorFink, Michael J.
dc.creatorBreslmayr, Erik
dc.creatorLaurent, Christophe
dc.creatorFeroz, Saima
dc.creatorGoncalves, Leticia C. P.
dc.creatorRial, Daniela V.
dc.creatorMihovilovic, Marko D.
dc.creatorBommarius, Andreas S.
dc.creatorLudwig, Roland
dc.creatorOostenbrink, Chris
dc.creatorRudroff, Florian
dc.date.accessioned2025-02-04T16:31:56Z
dc.date.available2025-02-04T16:31:56Z
dc.date.issued2022-09-13
dc.description.abstractThe 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.
dc.description.filFil: Mansouri, Hamid R. Institute of Applied Synthetic Chemistry; Austria.
dc.description.filFil: Gracia Carmona, Oriol. University of Natural Resources and Life Science. Institute of Molecular Modeling and Simulation; Austria.
dc.description.filFil: Jodlbauer, Julia. Institute of Applied Synthetic Chemistry: Austria.
dc.description.filFil: Schweiger, Lorenz. University of Natural Resources and Life Sciences. Biocatalysis and Biosensing Laboratory. Department of Food Science and Technology; Austria.
dc.description.filFil: Fink, Michael J. Institute of Applied Synthetic Chemistry; Austria.
dc.description.filFil: Breslmayr, Erik. University of Natural Resources and Life Sciences. Biocatalysis and Biosensing Laboratory. Department of Food Science and Technology; Austria.
dc.description.filFil: Laurent, Christophe. University of Natural Resources and Life Sciences Biocatalysis and Biosensing Laboratory. Department of Food Science and Technology; Austria.
dc.description.filFil: Feroz, Saima. Institute of Applied Synthetic Chemistry; Austria.
dc.description.filFil: Feroz, Saima. University of Hafr Al Batin. College of Science. Department of Biosciences.; Saudi Arabia.
dc.description.filFil: Goncalves, Leticia C. P. Université Côte d’Azur. Institut de Chimie de Nice; France.
dc.description.filFil: Rial, Daniela V. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Ciencias Biológicas. Área Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); Argentina.
dc.description.filFil: Mihovilovic, Marko D. Institute of Applied Synthetic Chemistry; Austria.
dc.description.filFil: Bommarius, Andreas S. Georgia Institute of Technology. School of Chemical & Biomolecular Engineering, Engineered Biosystems Building; United States.
dc.description.filFil: Ludwig, Roland. University of Natural Resources and Life Sciences. Biocatalysis and Biosensing Laboratory. Department of Food Science and Technology; Austria.
dc.description.filFil: Oostenbrink, Chris. University of Natural Resources and Life Science. Institute of Molecular Modeling and Simulation; Austria.
dc.description.filFil: Rudroff, Florian. Institute of Applied Synthetic Chemistry; Austria.
dc.description.sponsorshipTU Wien
dc.description.sponsorshipABC-Top Anschubfinanzierung
dc.description.sponsorshipAustrian agency for international cooperation in education and research
dc.description.sponsorshipOxyGreen: 212281
dc.description.sponsorshipAustrian Science Fund: W1224, M1948-N28, P-18945
dc.description.sponsorshipConsejo Nacional de Investigaciones Científicas y Técnicas (CONICET)
dc.description.sponsorshipUniversidad Nacional de Rosario (UNR)
dc.description.versionpeerreviewed
dc.format.extent11761-11766
dc.identifier.issn2155-5435
dc.identifier.urihttps://hdl.handle.net/2133/28785
dc.language.isoen
dc.publisherAmerican Chemical Society
dc.relation.publisherversionhttps://pubs.acs.org/doi/10.1021/acscatal.2c03225
dc.relation.publisherversionhttps://doi.org/10.1021/acscatal.2c03225
dc.rightsopenAccess
dc.rights.holderMansouri, Hamid R.
dc.rights.holderGracia Carmona, Oriol
dc.rights.holderJodlbauer, Julia
dc.rights.holderSchweiger, Lorenz
dc.rights.holderFink, Michael J.
dc.rights.holderBreslmayr, Erik
dc.rights.holderLaurent, Christophe
dc.rights.holderFeroz, Saima
dc.rights.holderGoncalves, Leticia C. P.
dc.rights.holderRial, Daniela V.
dc.rights.holderMihovilovic, Marko D.
dc.rights.holderBommarius, Andreas S.
dc.rights.holderLudwig, Roland
dc.rights.holderOostenbrink, Chris
dc.rights.holderRudroff, Florian
dc.rights.holderUniversidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas
dc.rights.textAttribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/
dc.subjectProtein engineering
dc.subjectEnzyme stabilization
dc.subjectCyclohexanone monooxygenase
dc.subjectStructure guided consensus approach
dc.subjectOxidation
dc.subjectMutagenesis
dc.titleMutations increasing cofactor affinity, improve stability and activity of a Baeyer Villiger monooxygenase
dc.typearticulo
dc.type.collectionarticulo
dc.type.versionpublishedVersion

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