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

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
Año de publicación
2022
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
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.
Fil: Mansouri, Hamid R.. Vienna University of Technology; Austria
Fil: Gracia Carmona, Oriol. Universitat Fur Bodenkultur Wien; Austria
Fil: Jodlbauer, Julia. Vienna University of Technology; Austria
Fil: Schweiger, Lorenz. Universitat Fur Bodenkultur Wien; Austria
Fil: Fink, Michael J.. Vienna University of Technology; Austria
Fil: Breslmayr, Erik. Universitat Fur Bodenkultur Wien; Austria
Fil: Laurent, Christophe. Universitat Fur Bodenkultur Wien; Austria
Fil: Feroz, Saima. Vienna University of Technology; Austria
Fil: Goncalves, Leticia C. P.. Université Côte D'azur; Francia
Fil: Rial, Daniela Veronica. Universidad Nacional de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina
Fil: Mihovilovic, Marko D.. Vienna University of Technology; Austria
Fil: Bommarius, Andreas S.. Georgia Institute of Techology; Estados Unidos
Fil: Ludwig, Roland. Universitat Fur Bodenkultur Wien; Austria
Fil: Oostenbrink, Chris. Universitat Fur Bodenkultur Wien; Austria
Fil: Rudroff, Florian. Vienna University of Technology; Austria
Materia
CYCLOHEXANONE MONOOXYGENASE
ENZYME STABILIZATION
MUTAGENESIS
OXIDATION
PROTEIN ENGINEERING
STRUCTURE-GUIDED CONSENSUS APPROACH
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/218216
Acceder
id CONICETDig_f762b18ea07cb55a3194c32987e1ff57
oai_identifier_str oai:ri.conicet.gov.ar:11336/218216
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Mutations Increasing Cofactor Affinity, Improve Stability and Activity of a Baeyer-Villiger MonooxygenaseMansouri, Hamid R.Gracia Carmona, OriolJodlbauer, JuliaSchweiger, LorenzFink, Michael J.Breslmayr, ErikLaurent, ChristopheFeroz, SaimaGoncalves, Leticia C. P.Rial, Daniela VeronicaMihovilovic, Marko D.Bommarius, Andreas S.Ludwig, RolandOostenbrink, ChrisRudroff, FlorianCYCLOHEXANONE MONOOXYGENASEENZYME STABILIZATIONMUTAGENESISOXIDATIONPROTEIN ENGINEERINGSTRUCTURE-GUIDED CONSENSUS APPROACHhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1The 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.Fil: Mansouri, Hamid R.. Vienna University of Technology; AustriaFil: Gracia Carmona, Oriol. Universitat Fur Bodenkultur Wien; AustriaFil: Jodlbauer, Julia. Vienna University of Technology; AustriaFil: Schweiger, Lorenz. Universitat Fur Bodenkultur Wien; AustriaFil: Fink, Michael J.. Vienna University of Technology; AustriaFil: Breslmayr, Erik. Universitat Fur Bodenkultur Wien; AustriaFil: Laurent, Christophe. Universitat Fur Bodenkultur Wien; AustriaFil: Feroz, Saima. Vienna University of Technology; AustriaFil: Goncalves, Leticia C. P.. Université Côte D'azur; FranciaFil: Rial, Daniela Veronica. Universidad Nacional de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Mihovilovic, Marko D.. Vienna University of Technology; AustriaFil: Bommarius, Andreas S.. Georgia Institute of Techology; Estados UnidosFil: Ludwig, Roland. Universitat Fur Bodenkultur Wien; AustriaFil: Oostenbrink, Chris. Universitat Fur Bodenkultur Wien; AustriaFil: Rudroff, Florian. Vienna University of Technology; AustriaAmerican Chemical Society2022-09info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/218216Mansouri, Hamid R.; Gracia Carmona, Oriol; Jodlbauer, Julia; Schweiger, Lorenz; Fink, Michael J.; et al.; Mutations Increasing Cofactor Affinity, Improve Stability and Activity of a Baeyer-Villiger Monooxygenase; American Chemical Society; ACS Catalysis; 12; 19; 9-2022; 11761-117662155-5435CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1021/acscatal.2c03225info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-15T15:41:20Zoai:ri.conicet.gov.ar:11336/218216instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-10-15 15:41:20.481CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Mutations Increasing Cofactor Affinity, Improve Stability and Activity of a Baeyer-Villiger Monooxygenase
title Mutations Increasing Cofactor Affinity, Improve Stability and Activity of a Baeyer-Villiger Monooxygenase
spellingShingle Mutations Increasing Cofactor Affinity, Improve Stability and Activity of a Baeyer-Villiger Monooxygenase
Mansouri, Hamid R.
CYCLOHEXANONE MONOOXYGENASE
ENZYME STABILIZATION
MUTAGENESIS
OXIDATION
PROTEIN ENGINEERING
STRUCTURE-GUIDED CONSENSUS APPROACH
title_short Mutations Increasing Cofactor Affinity, Improve Stability and Activity of a Baeyer-Villiger Monooxygenase
title_full Mutations Increasing Cofactor Affinity, Improve Stability and Activity of a Baeyer-Villiger Monooxygenase
title_fullStr Mutations Increasing Cofactor Affinity, Improve Stability and Activity of a Baeyer-Villiger Monooxygenase
title_full_unstemmed Mutations Increasing Cofactor Affinity, Improve Stability and Activity of a Baeyer-Villiger Monooxygenase
title_sort Mutations Increasing Cofactor Affinity, Improve Stability and Activity of a Baeyer-Villiger Monooxygenase
dc.creator.none.fl_str_mv 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
author Mansouri, Hamid R.
author_facet 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
author_role author
author2 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
author2_role author
author
author
author
author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv CYCLOHEXANONE MONOOXYGENASE
ENZYME STABILIZATION
MUTAGENESIS
OXIDATION
PROTEIN ENGINEERING
STRUCTURE-GUIDED CONSENSUS APPROACH
topic CYCLOHEXANONE MONOOXYGENASE
ENZYME STABILIZATION
MUTAGENESIS
OXIDATION
PROTEIN ENGINEERING
STRUCTURE-GUIDED CONSENSUS APPROACH
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv 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.
Fil: Mansouri, Hamid R.. Vienna University of Technology; Austria
Fil: Gracia Carmona, Oriol. Universitat Fur Bodenkultur Wien; Austria
Fil: Jodlbauer, Julia. Vienna University of Technology; Austria
Fil: Schweiger, Lorenz. Universitat Fur Bodenkultur Wien; Austria
Fil: Fink, Michael J.. Vienna University of Technology; Austria
Fil: Breslmayr, Erik. Universitat Fur Bodenkultur Wien; Austria
Fil: Laurent, Christophe. Universitat Fur Bodenkultur Wien; Austria
Fil: Feroz, Saima. Vienna University of Technology; Austria
Fil: Goncalves, Leticia C. P.. Université Côte D'azur; Francia
Fil: Rial, Daniela Veronica. Universidad Nacional de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina
Fil: Mihovilovic, Marko D.. Vienna University of Technology; Austria
Fil: Bommarius, Andreas S.. Georgia Institute of Techology; Estados Unidos
Fil: Ludwig, Roland. Universitat Fur Bodenkultur Wien; Austria
Fil: Oostenbrink, Chris. Universitat Fur Bodenkultur Wien; Austria
Fil: Rudroff, Florian. Vienna University of Technology; Austria
description 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.
publishDate 2022
dc.date.none.fl_str_mv 2022-09
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/218216
Mansouri, Hamid R.; Gracia Carmona, Oriol; Jodlbauer, Julia; Schweiger, Lorenz; Fink, Michael J.; et al.; Mutations Increasing Cofactor Affinity, Improve Stability and Activity of a Baeyer-Villiger Monooxygenase; American Chemical Society; ACS Catalysis; 12; 19; 9-2022; 11761-11766
2155-5435
CONICET Digital
CONICET
url http://hdl.handle.net/11336/218216
identifier_str_mv Mansouri, Hamid R.; Gracia Carmona, Oriol; Jodlbauer, Julia; Schweiger, Lorenz; Fink, Michael J.; et al.; Mutations Increasing Cofactor Affinity, Improve Stability and Activity of a Baeyer-Villiger Monooxygenase; American Chemical Society; ACS Catalysis; 12; 19; 9-2022; 11761-11766
2155-5435
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1021/acscatal.2c03225
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv American Chemical Society
publisher.none.fl_str_mv American Chemical Society
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
_version_ 1846083524755456000
score 13.22299

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