Cryo-EM structure of GH43 β-Xylosidase from Enterobacter cloacae provides insights into substrate specificity and the role of an auxiliary domain in enzymatic activity

Autores
Briganti, Lorenzo; Godoy, Andre Schutzer; Capetti, Caio; Fernandes, Rafaela Sachetto; Pellegrini, Vanessa O. A.; Ontañon, Ornella Mailen; Campos, Eleonora; Portugal, Rodrigo V.; Polikarpov, Igor
Año de publicación
2026
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Xylan is the second most abundant polysaccharide in plant cell walls, consisting of a β-1,4-linked xylopyranosyl backbone that can be substituted with various side chains. Depolymerization of xylan is predominantly catalyzed by the coordinated activity of β-xylanases and β-xylosidases. In this study, we determined the cryo-electron microscopy (cryo-EM) structure of Enterobacter cloacae β-xylosidase (EcXyl43), a glycoside hydrolase family 43 (GH43) enzyme. Additionally, we resolved the X-ray crystal structure of a catalytically inactive F507A mutant of EcXyl43. Together, these structures represent the first structural characterization of a β-xylosidase from the Enterobacter genus using both X-ray diffraction and cryoEM. Furthermore, to elucidate the molecular basis of substrate recognition and specificity, we conducted molecular dynamics simulations of the enzyme. Structural and computational analysis of EcXyl43 identified key determinants of the enzyme's preference for longer xylooligosaccharides and revealed how noncatalytic residues within the auxiliary domain modulate its activity.
Instituto de Biotecnología
Fil: Briganti, Lorenzo. University of São Paulo. São Carlos Institute of Physics; Brasil
Fil: Godoy, Andre Schutzer. University of São Paulo. São Carlos Institute of Physics; Brasil
Fil: Godoy, Andre Schutzer. Echo Life Sciences; Brasil
Fil: Capetti, Caio. University of São Paulo. São Carlos Institute of Physics; Brasil
Fil: Fernandes, Rafaela Sachetto. University of São Paulo. São Carlos Institute of Physics; Brasil
Fil: Pellegrini, Vanessa O. A. University of São Paulo. São Carlos Institute of Physics; Brasil
Fil: Ontañon, Ornella Mailen. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina
Fil: Ontañon, Ornella Mailen. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Campos, Eleonora. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina
Fil: Campos, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Portugal, Rodrigo V. Brazilian Center for Research in Energy and Materials (CNPEM). Brazilian Nanotechnology National Laboratory; Brasil
Fil: Polikarpov, Igor. University of São Paulo. São Carlos Institute of Physics; Brasil
Fuente
The FEBS Journal (First published: 18 May 2026)
Materia
Xylans
Enzyme Activity
Xilano
Enterobacter cloacae
Actividad Enzimática
Nivel de accesibilidad
acceso abierto
Condiciones de uso
http://creativecommons.org/licenses/by-nc-sa/4.0/
Repositorio
INTA Digital (INTA)
Institución
Instituto Nacional de Tecnología Agropecuaria
OAI Identificador
oai:localhost:20.500.12123/26270
Acceder
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oai_identifier_str oai:localhost:20.500.12123/26270
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network_name_str INTA Digital (INTA)
spelling Cryo-EM structure of GH43 β-Xylosidase from Enterobacter cloacae provides insights into substrate specificity and the role of an auxiliary domain in enzymatic activityBriganti, LorenzoGodoy, Andre SchutzerCapetti, CaioFernandes, Rafaela SachettoPellegrini, Vanessa O. A.Ontañon, Ornella MailenCampos, EleonoraPortugal, Rodrigo V.Polikarpov, IgorXylansEnzyme ActivityXilanoEnterobacter cloacaeActividad EnzimáticaXylan is the second most abundant polysaccharide in plant cell walls, consisting of a β-1,4-linked xylopyranosyl backbone that can be substituted with various side chains. Depolymerization of xylan is predominantly catalyzed by the coordinated activity of β-xylanases and β-xylosidases. In this study, we determined the cryo-electron microscopy (cryo-EM) structure of Enterobacter cloacae β-xylosidase (EcXyl43), a glycoside hydrolase family 43 (GH43) enzyme. Additionally, we resolved the X-ray crystal structure of a catalytically inactive F507A mutant of EcXyl43. Together, these structures represent the first structural characterization of a β-xylosidase from the Enterobacter genus using both X-ray diffraction and cryoEM. Furthermore, to elucidate the molecular basis of substrate recognition and specificity, we conducted molecular dynamics simulations of the enzyme. Structural and computational analysis of EcXyl43 identified key determinants of the enzyme's preference for longer xylooligosaccharides and revealed how noncatalytic residues within the auxiliary domain modulate its activity.Instituto de BiotecnologíaFil: Briganti, Lorenzo. University of São Paulo. São Carlos Institute of Physics; BrasilFil: Godoy, Andre Schutzer. University of São Paulo. São Carlos Institute of Physics; BrasilFil: Godoy, Andre Schutzer. Echo Life Sciences; BrasilFil: Capetti, Caio. University of São Paulo. São Carlos Institute of Physics; BrasilFil: Fernandes, Rafaela Sachetto. University of São Paulo. São Carlos Institute of Physics; BrasilFil: Pellegrini, Vanessa O. A. University of São Paulo. São Carlos Institute of Physics; BrasilFil: Ontañon, Ornella Mailen. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Ontañon, Ornella Mailen. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Campos, Eleonora. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Campos, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Portugal, Rodrigo V. Brazilian Center for Research in Energy and Materials (CNPEM). Brazilian Nanotechnology National Laboratory; BrasilFil: Polikarpov, Igor. University of São Paulo. São Carlos Institute of Physics; BrasilWiley2026-05-20T10:08:02Z2026-05-20T10:08:02Z2026-05info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfhttp://hdl.handle.net/20.500.12123/26270https://febs.onlinelibrary.wiley.com/doi/10.1111/febs.705901742-4658https://doi.org/10.1111/febs.70590The FEBS Journal (First published: 18 May 2026)reponame:INTA Digital (INTA)instname:Instituto Nacional de Tecnología Agropecuariaenginfo:eu-repo/semantics/openAccesshttp://creativecommons.org/licenses/by-nc-sa/4.0/Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)2026-05-28T08:47:27Zoai:localhost:20.500.12123/26270instacron:INTAInstitucionalhttp://repositorio.inta.gob.ar/Organismo científico-tecnológicoNo correspondehttp://repositorio.inta.gob.ar/oai/requesttripaldi.nicolas@inta.gob.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:l2026-05-28 08:47:27.546INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuariafalse
dc.title.none.fl_str_mv Cryo-EM structure of GH43 β-Xylosidase from Enterobacter cloacae provides insights into substrate specificity and the role of an auxiliary domain in enzymatic activity
title Cryo-EM structure of GH43 β-Xylosidase from Enterobacter cloacae provides insights into substrate specificity and the role of an auxiliary domain in enzymatic activity
spellingShingle Cryo-EM structure of GH43 β-Xylosidase from Enterobacter cloacae provides insights into substrate specificity and the role of an auxiliary domain in enzymatic activity
Briganti, Lorenzo
Xylans
Enzyme Activity
Xilano
Enterobacter cloacae
Actividad Enzimática
title_short Cryo-EM structure of GH43 β-Xylosidase from Enterobacter cloacae provides insights into substrate specificity and the role of an auxiliary domain in enzymatic activity
title_full Cryo-EM structure of GH43 β-Xylosidase from Enterobacter cloacae provides insights into substrate specificity and the role of an auxiliary domain in enzymatic activity
title_fullStr Cryo-EM structure of GH43 β-Xylosidase from Enterobacter cloacae provides insights into substrate specificity and the role of an auxiliary domain in enzymatic activity
title_full_unstemmed Cryo-EM structure of GH43 β-Xylosidase from Enterobacter cloacae provides insights into substrate specificity and the role of an auxiliary domain in enzymatic activity
title_sort Cryo-EM structure of GH43 β-Xylosidase from Enterobacter cloacae provides insights into substrate specificity and the role of an auxiliary domain in enzymatic activity
dc.creator.none.fl_str_mv Briganti, Lorenzo
Godoy, Andre Schutzer
Capetti, Caio
Fernandes, Rafaela Sachetto
Pellegrini, Vanessa O. A.
Ontañon, Ornella Mailen
Campos, Eleonora
Portugal, Rodrigo V.
Polikarpov, Igor
author Briganti, Lorenzo
author_facet Briganti, Lorenzo
Godoy, Andre Schutzer
Capetti, Caio
Fernandes, Rafaela Sachetto
Pellegrini, Vanessa O. A.
Ontañon, Ornella Mailen
Campos, Eleonora
Portugal, Rodrigo V.
Polikarpov, Igor
author_role author
author2 Godoy, Andre Schutzer
Capetti, Caio
Fernandes, Rafaela Sachetto
Pellegrini, Vanessa O. A.
Ontañon, Ornella Mailen
Campos, Eleonora
Portugal, Rodrigo V.
Polikarpov, Igor
author2_role author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Xylans
Enzyme Activity
Xilano
Enterobacter cloacae
Actividad Enzimática
topic Xylans
Enzyme Activity
Xilano
Enterobacter cloacae
Actividad Enzimática
dc.description.none.fl_txt_mv Xylan is the second most abundant polysaccharide in plant cell walls, consisting of a β-1,4-linked xylopyranosyl backbone that can be substituted with various side chains. Depolymerization of xylan is predominantly catalyzed by the coordinated activity of β-xylanases and β-xylosidases. In this study, we determined the cryo-electron microscopy (cryo-EM) structure of Enterobacter cloacae β-xylosidase (EcXyl43), a glycoside hydrolase family 43 (GH43) enzyme. Additionally, we resolved the X-ray crystal structure of a catalytically inactive F507A mutant of EcXyl43. Together, these structures represent the first structural characterization of a β-xylosidase from the Enterobacter genus using both X-ray diffraction and cryoEM. Furthermore, to elucidate the molecular basis of substrate recognition and specificity, we conducted molecular dynamics simulations of the enzyme. Structural and computational analysis of EcXyl43 identified key determinants of the enzyme's preference for longer xylooligosaccharides and revealed how noncatalytic residues within the auxiliary domain modulate its activity.
Instituto de Biotecnología
Fil: Briganti, Lorenzo. University of São Paulo. São Carlos Institute of Physics; Brasil
Fil: Godoy, Andre Schutzer. University of São Paulo. São Carlos Institute of Physics; Brasil
Fil: Godoy, Andre Schutzer. Echo Life Sciences; Brasil
Fil: Capetti, Caio. University of São Paulo. São Carlos Institute of Physics; Brasil
Fil: Fernandes, Rafaela Sachetto. University of São Paulo. São Carlos Institute of Physics; Brasil
Fil: Pellegrini, Vanessa O. A. University of São Paulo. São Carlos Institute of Physics; Brasil
Fil: Ontañon, Ornella Mailen. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina
Fil: Ontañon, Ornella Mailen. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Campos, Eleonora. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina
Fil: Campos, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Portugal, Rodrigo V. Brazilian Center for Research in Energy and Materials (CNPEM). Brazilian Nanotechnology National Laboratory; Brasil
Fil: Polikarpov, Igor. University of São Paulo. São Carlos Institute of Physics; Brasil
description Xylan is the second most abundant polysaccharide in plant cell walls, consisting of a β-1,4-linked xylopyranosyl backbone that can be substituted with various side chains. Depolymerization of xylan is predominantly catalyzed by the coordinated activity of β-xylanases and β-xylosidases. In this study, we determined the cryo-electron microscopy (cryo-EM) structure of Enterobacter cloacae β-xylosidase (EcXyl43), a glycoside hydrolase family 43 (GH43) enzyme. Additionally, we resolved the X-ray crystal structure of a catalytically inactive F507A mutant of EcXyl43. Together, these structures represent the first structural characterization of a β-xylosidase from the Enterobacter genus using both X-ray diffraction and cryoEM. Furthermore, to elucidate the molecular basis of substrate recognition and specificity, we conducted molecular dynamics simulations of the enzyme. Structural and computational analysis of EcXyl43 identified key determinants of the enzyme's preference for longer xylooligosaccharides and revealed how noncatalytic residues within the auxiliary domain modulate its activity.
publishDate 2026
dc.date.none.fl_str_mv 2026-05-20T10:08:02Z
2026-05-20T10:08:02Z
2026-05
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/20.500.12123/26270
https://febs.onlinelibrary.wiley.com/doi/10.1111/febs.70590
1742-4658
https://doi.org/10.1111/febs.70590
url http://hdl.handle.net/20.500.12123/26270
https://febs.onlinelibrary.wiley.com/doi/10.1111/febs.70590
https://doi.org/10.1111/febs.70590
identifier_str_mv 1742-4658
dc.language.none.fl_str_mv eng
language eng
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
http://creativecommons.org/licenses/by-nc-sa/4.0/
Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)
eu_rights_str_mv openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by-nc-sa/4.0/
Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Wiley
publisher.none.fl_str_mv Wiley
dc.source.none.fl_str_mv The FEBS Journal (First published: 18 May 2026)
reponame:INTA Digital (INTA)
instname:Instituto Nacional de Tecnología Agropecuaria
reponame_str INTA Digital (INTA)
collection INTA Digital (INTA)
instname_str Instituto Nacional de Tecnología Agropecuaria
repository.name.fl_str_mv INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuaria
repository.mail.fl_str_mv tripaldi.nicolas@inta.gob.ar
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score 13.143419

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