Multicopper oxidases with laccase-ferroxidase activity: Classification and study of ferroxidase activity determinants in a member from Heterobasidion annosum s. l

Multi-copper oxidases (MCO) share a common molecular architecture and the use of copper ions as cofactors to reduce O2 to H2O, but show high sequence heterogeneity and functional diversity. Many new emerging MCO genes are wrongly annotated as laccases, the largest group of MCOs, with the widest rang...

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Autores: Aza, Pablo, Molpeceres, Gonzalo, Vind, Jesper, Camarero, Susana
Tipo de documento: artigo
Estado:Versão publicada
Data de publicação:2023
País:España
Recursos:Consejo Superior de Investigaciones Científicas (CSIC)
Repositório:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:dnet:digitalcsic_::1e19acf3fab08c389b8162f4935aebcf
Acesso em linha:http://hdl.handle.net/10261/427549
https://digital.csic.es/handle/10261/288613
Access Level:Acceso aberto
Palavra-chave:Heterologous expression
Laccase-ferroxidase
Multicopper oxidases
Phylogenetic analysis
Structure-function
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dc.title.none.fl_str_mv Multicopper oxidases with laccase-ferroxidase activity: Classification and study of ferroxidase activity determinants in a member from Heterobasidion annosum s. l
title Multicopper oxidases with laccase-ferroxidase activity: Classification and study of ferroxidase activity determinants in a member from Heterobasidion annosum s. l
spellingShingle Multicopper oxidases with laccase-ferroxidase activity: Classification and study of ferroxidase activity determinants in a member from Heterobasidion annosum s. l
Aza, Pablo
Heterologous expression
Laccase-ferroxidase
Multicopper oxidases
Phylogenetic analysis
Structure-function
title_short Multicopper oxidases with laccase-ferroxidase activity: Classification and study of ferroxidase activity determinants in a member from Heterobasidion annosum s. l
title_full Multicopper oxidases with laccase-ferroxidase activity: Classification and study of ferroxidase activity determinants in a member from Heterobasidion annosum s. l
title_fullStr Multicopper oxidases with laccase-ferroxidase activity: Classification and study of ferroxidase activity determinants in a member from Heterobasidion annosum s. l
title_full_unstemmed Multicopper oxidases with laccase-ferroxidase activity: Classification and study of ferroxidase activity determinants in a member from Heterobasidion annosum s. l
title_sort Multicopper oxidases with laccase-ferroxidase activity: Classification and study of ferroxidase activity determinants in a member from Heterobasidion annosum s. l
dc.creator.none.fl_str_mv Aza, Pablo
Molpeceres, Gonzalo
Vind, Jesper
Camarero, Susana
author Aza, Pablo
author_facet Aza, Pablo
Molpeceres, Gonzalo
Vind, Jesper
Camarero, Susana
author_role author
author2 Molpeceres, Gonzalo
Vind, Jesper
Camarero, Susana
author2_role author
author
author
dc.contributor.none.fl_str_mv Bio-based Industries Joint Undertaking
European Commission
Consejo Superior de Investigaciones Científicas (España)
Ministerio de Ciencia, Innovación y Universidades (España)
Novozymes
Molpeceres, Gonzalo [0000-0002-4366-9412]
Camarero, Susana [0000-0002-2812-895X]
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Heterologous expression
Laccase-ferroxidase
Multicopper oxidases
Phylogenetic analysis
Structure-function
topic Heterologous expression
Laccase-ferroxidase
Multicopper oxidases
Phylogenetic analysis
Structure-function
description Multi-copper oxidases (MCO) share a common molecular architecture and the use of copper ions as cofactors to reduce O2 to H2O, but show high sequence heterogeneity and functional diversity. Many new emerging MCO genes are wrongly annotated as laccases, the largest group of MCOs, with the widest range of biotechnological applications (particularly those from basidiomycete fungi) due to their ability to oxidise aromatic compounds and lignin. Thus, comprehensive studies for a better classification and structure-function characterisation of MCO families are required. Laccase-ferroxidases (LAC-FOXs) constitute a separate and unexplored group of MCOs with proposed dual features between laccases and ferroxidases. We aim to better define this cluster and the structural determinants underlying putative hybrid activity. We performed a phylogenetic analysis of the LAC-FOXs from basidiomycete fungi, that resulted in two subgroups. This division seemed to correlate with the presence or absence of some of the three acidic residues responsible for ferroxidase activity in Fet3p from Saccharomyces cerevisiae. One of these LAC-FOXs (with only one of these residues) from the fungus Heterobasidion annosum s. l. (HaLF) was synthesised, heterologously expressed and characterised to evaluate its catalytic activity. HaLF oxidised typical laccase substrates (phenols, aryl amines and N-heterocycles), but no Fe (II). The enzyme was subjected to site-directed mutagenesis to determine the key residues that confer ferroxidase activity. The mutated HaLF variant with full restoration of the three acidic residues exhibited efficient ferroxidase activity, while it partially retained the wide-range oxidative activity of the native enzyme associated to laccases sensu stricto.
publishDate 2023
dc.date.none.fl_str_mv 2023
2026
2026
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/427549
https://digital.csic.es/handle/10261/288613
url http://hdl.handle.net/10261/427549
https://digital.csic.es/handle/10261/288613
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv #PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
GA 792070
info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BIO2017-86559-R
info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2021-2023/PID2021-126384OB-I00
https://doi.org/10.1016/j.csbj.2023.01.030
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv American Association for the Advancement of Science
publisher.none.fl_str_mv American Association for the Advancement of Science
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
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repository.name.fl_str_mv
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spelling Multicopper oxidases with laccase-ferroxidase activity: Classification and study of ferroxidase activity determinants in a member from Heterobasidion annosum s. lAza, PabloMolpeceres, GonzaloVind, JesperCamarero, SusanaHeterologous expressionLaccase-ferroxidaseMulticopper oxidasesPhylogenetic analysisStructure-functionMulti-copper oxidases (MCO) share a common molecular architecture and the use of copper ions as cofactors to reduce O2 to H2O, but show high sequence heterogeneity and functional diversity. Many new emerging MCO genes are wrongly annotated as laccases, the largest group of MCOs, with the widest range of biotechnological applications (particularly those from basidiomycete fungi) due to their ability to oxidise aromatic compounds and lignin. Thus, comprehensive studies for a better classification and structure-function characterisation of MCO families are required. Laccase-ferroxidases (LAC-FOXs) constitute a separate and unexplored group of MCOs with proposed dual features between laccases and ferroxidases. We aim to better define this cluster and the structural determinants underlying putative hybrid activity. We performed a phylogenetic analysis of the LAC-FOXs from basidiomycete fungi, that resulted in two subgroups. This division seemed to correlate with the presence or absence of some of the three acidic residues responsible for ferroxidase activity in Fet3p from Saccharomyces cerevisiae. One of these LAC-FOXs (with only one of these residues) from the fungus Heterobasidion annosum s. l. (HaLF) was synthesised, heterologously expressed and characterised to evaluate its catalytic activity. HaLF oxidised typical laccase substrates (phenols, aryl amines and N-heterocycles), but no Fe (II). The enzyme was subjected to site-directed mutagenesis to determine the key residues that confer ferroxidase activity. The mutated HaLF variant with full restoration of the three acidic residues exhibited efficient ferroxidase activity, while it partially retained the wide-range oxidative activity of the native enzyme associated to laccases sensu stricto.This work was supported by WoodZymes project funded by the Bio-based Industries Joint Undertaking (BBI JU) under GA 792070. The BBI JU received support from the EU’s H2020 research and innovation programme and the Bio Based Industries Consortium. The work was also supported by GENOBIOREF project funded by MINECO/FEDER (BIO2017-86559-R), the LIG2PLAST project funded by MCIN/AEI/FEDER (PID2021-126384OB-I00), and the CSIC Interdisciplinary Platform for Sustainable Plastics towards a Circular Economy (PTI-SusPlast+). P. Aza acknowledges an FPU grant from the Spanish Ministry of Universities including the grant for his short-term stay at Novozymes. G. Molpeceres thanks the Tatiana Pérez de Guzman el Bueno Foundation for his predoctoral environment grant.Peer reviewedAmerican Association for the Advancement of ScienceBio-based Industries Joint UndertakingEuropean CommissionConsejo Superior de Investigaciones Científicas (España)Ministerio de Ciencia, Innovación y Universidades (España)NovozymesMolpeceres, Gonzalo [0000-0002-4366-9412]Camarero, Susana [0000-0002-2812-895X]Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]202620262023info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionapplication/pdfhttp://hdl.handle.net/10261/427549https://digital.csic.es/handle/10261/288613reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE#GA 792070info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BIO2017-86559-Rinfo:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2021-2023/PID2021-126384OB-I00https://doi.org/10.1016/j.csbj.2023.01.030Síinfo:eu-repo/semantics/openAccessoai:dnet:digitalcsic_::1e19acf3fab08c389b8162f4935aebcf2026-05-22T06:33:51Z
score 15.811543

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