Embedding a feruloyl esterase active site into a thermophilic endoxylanase scaffold for the degradation of feruloylated xylans

The structural complexity of xylan makes its complete degradation challenging. Strategies to improve its hydrolysis often requires enzyme cocktails with multiple specific activities or proteins harboring multiple catalytic domains. Here, we introduce a novel approach through the design of Xyn11m1, a...

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Autores: Muñoz Tafalla, Rubén, Cea Rama, Isabel, Cervantes, Fadia V., González-Alfonso, José L., Plou Gasca, Francisco José, Polaina, Julio, Sanz-Aparicio, J., Ferrer, Manuel, Guallar, Víctor, Talens Perales, David
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2025
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/401526
Acceso en línea:http://hdl.handle.net/10261/401526
https://api.elsevier.com/content/abstract/scopus_id/105015144868
Access Level:acceso abierto
Palabra clave:Feruloyl esterase
PluriZyme
Protein Engineering
Xylan
Xylanase
proteins
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network_acronym_str ES
network_name_str España
repository_id_str
dc.title.none.fl_str_mv Embedding a feruloyl esterase active site into a thermophilic endoxylanase scaffold for the degradation of feruloylated xylans
title Embedding a feruloyl esterase active site into a thermophilic endoxylanase scaffold for the degradation of feruloylated xylans
spellingShingle Embedding a feruloyl esterase active site into a thermophilic endoxylanase scaffold for the degradation of feruloylated xylans
Muñoz Tafalla, Rubén
Feruloyl esterase
PluriZyme
Protein Engineering
Xylan
Xylanase
proteins
title_short Embedding a feruloyl esterase active site into a thermophilic endoxylanase scaffold for the degradation of feruloylated xylans
title_full Embedding a feruloyl esterase active site into a thermophilic endoxylanase scaffold for the degradation of feruloylated xylans
title_fullStr Embedding a feruloyl esterase active site into a thermophilic endoxylanase scaffold for the degradation of feruloylated xylans
title_full_unstemmed Embedding a feruloyl esterase active site into a thermophilic endoxylanase scaffold for the degradation of feruloylated xylans
title_sort Embedding a feruloyl esterase active site into a thermophilic endoxylanase scaffold for the degradation of feruloylated xylans
dc.creator.none.fl_str_mv Muñoz Tafalla, Rubén
Cea Rama, Isabel
Cervantes, Fadia V.
González-Alfonso, José L.
Plou Gasca, Francisco José
Polaina, Julio
Sanz-Aparicio, J.
Ferrer, Manuel
Guallar, Víctor
Talens Perales, David
author Muñoz Tafalla, Rubén
author_facet Muñoz Tafalla, Rubén
Cea Rama, Isabel
Cervantes, Fadia V.
González-Alfonso, José L.
Plou Gasca, Francisco José
Polaina, Julio
Sanz-Aparicio, J.
Ferrer, Manuel
Guallar, Víctor
Talens Perales, David
author_role author
author2 Cea Rama, Isabel
Cervantes, Fadia V.
González-Alfonso, José L.
Plou Gasca, Francisco José
Polaina, Julio
Sanz-Aparicio, J.
Ferrer, Manuel
Guallar, Víctor
Talens Perales, David
author2_role author
author
author
author
author
author
author
author
author
dc.contributor.none.fl_str_mv European Commission
Ministerio de Ciencia, Innovación y Universidades (España)
Agencia Estatal de Investigación (España)
Muñoz Tafalla, Rubén [0000-0003-4380-4888]
Cea Rama, Isabel [0000-0002-0929-2076]
Cervantes, Fadia V. [0000-0001-8844-8392]
González-Alfonso, José L. [0000-0002-3396-7985]
Plou, Francisco J. [0000-0003-0831-893X]
Polaina, Julio [0000-0001-9912-0640]
Sanz Aparicio, Julia [0000-0002-6849-8621]
Ferrer, Manuel [0000-0003-4962-4714]
Guallar, Víctor [0000-0002-4580-1114]
Talens Perales, David [0000-0002-8693-4239]
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Feruloyl esterase
PluriZyme
Protein Engineering
Xylan
Xylanase
proteins
topic Feruloyl esterase
PluriZyme
Protein Engineering
Xylan
Xylanase
proteins
description The structural complexity of xylan makes its complete degradation challenging. Strategies to improve its hydrolysis often requires enzyme cocktails with multiple specific activities or proteins harboring multiple catalytic domains. Here, we introduce a novel approach through the design of Xyn11m1, a multifunctional enzyme that combines endoxylanase and feruloyl esterase activities, two catalytic functions involved in the hydrolysis of feruloylated xylans. Using the PluriZyme concept, an artificial feruloyl esterase active site was engineered into the scaffold of a thermophilic glycoside hydrolase family 10 xylanase, Xyn11, from Pseudothermotoga thermarum. Computational design, guided by protein energy landscape exploration simulations, revealed a surface cavity that could accommodate feruloyl-L-arabinose and a xylopentaose (a 5-xylose xylan polymer) bearing a single feruloyl-L-arabinose substitution on the central xylose unit. This cavity was subsequently remodeled into a serine-histidine-aspartic/glutamic acid catalytic triad with feruloyl esterase activity. Molecular dynamics simulations confirmed the stability of the engineered active site. Xyn11m1 was successfully produced, crystallized, and characterized, and its xylanase activity at 90 °C against oat spelt xylan was comparable to that of the wild-type enzyme (713 ± 4 vs. 600 ± 8 units/mg), and it also displayed feruloyl esterase activity against methyl ferulate (140 ± 5 units/mg), a capability lacking in Xyn11. Notably, Xyn11m1 exhibited approximately 2.5-fold greater activity compared with Xyn11 (513 ± 27 vs. 222 ± 9 units/mg) against wheat bran xylan containing ferulic acid ester linked to arabinofuranosyl residues. This dual functionality enables efficient degradation of feruloylated xylans, highlighting the potential of PluriZymes to advance biomass deconstruction technologies.
publishDate 2025
dc.date.none.fl_str_mv 2025
2025
2025
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
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dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/401526
https://api.elsevier.com/content/abstract/scopus_id/105015144868
url http://hdl.handle.net/10261/401526
https://api.elsevier.com/content/abstract/scopus_id/105015144868
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
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Computational and structural biotechnology journal
The underlying dataset has been published at https://doi.org/10.5281/zenodo.15601971
https://doi.org/10.1016/j.csbj.2025.09.003
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dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
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instname_str Consejo Superior de Investigaciones Científicas (CSIC)
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spelling Embedding a feruloyl esterase active site into a thermophilic endoxylanase scaffold for the degradation of feruloylated xylansMuñoz Tafalla, RubénCea Rama, IsabelCervantes, Fadia V.González-Alfonso, José L.Plou Gasca, Francisco JoséPolaina, JulioSanz-Aparicio, J.Ferrer, ManuelGuallar, VíctorTalens Perales, DavidFeruloyl esterasePluriZymeProtein EngineeringXylanXylanaseproteinsThe structural complexity of xylan makes its complete degradation challenging. Strategies to improve its hydrolysis often requires enzyme cocktails with multiple specific activities or proteins harboring multiple catalytic domains. Here, we introduce a novel approach through the design of Xyn11m1, a multifunctional enzyme that combines endoxylanase and feruloyl esterase activities, two catalytic functions involved in the hydrolysis of feruloylated xylans. Using the PluriZyme concept, an artificial feruloyl esterase active site was engineered into the scaffold of a thermophilic glycoside hydrolase family 10 xylanase, Xyn11, from Pseudothermotoga thermarum. Computational design, guided by protein energy landscape exploration simulations, revealed a surface cavity that could accommodate feruloyl-L-arabinose and a xylopentaose (a 5-xylose xylan polymer) bearing a single feruloyl-L-arabinose substitution on the central xylose unit. This cavity was subsequently remodeled into a serine-histidine-aspartic/glutamic acid catalytic triad with feruloyl esterase activity. Molecular dynamics simulations confirmed the stability of the engineered active site. Xyn11m1 was successfully produced, crystallized, and characterized, and its xylanase activity at 90 °C against oat spelt xylan was comparable to that of the wild-type enzyme (713 ± 4 vs. 600 ± 8 units/mg), and it also displayed feruloyl esterase activity against methyl ferulate (140 ± 5 units/mg), a capability lacking in Xyn11. Notably, Xyn11m1 exhibited approximately 2.5-fold greater activity compared with Xyn11 (513 ± 27 vs. 222 ± 9 units/mg) against wheat bran xylan containing ferulic acid ester linked to arabinofuranosyl residues. This dual functionality enables efficient degradation of feruloylated xylans, highlighting the potential of PluriZymes to advance biomass deconstruction technologies.The authors (M.F., V.G.) thank the European Union’s Horizon 2020 Research and Innovation Programme for grant 101000327-FuturEnzyme and Horizon Europe for grant 101060625-Nymphe (M.F.). They also acknowledge funding from the Ministerio de Ciencia, Innovación y Universidades, Agencia Estatal de Investigación (AEI) (MICIU/AEI/10.13039/501100011033), FEDER, the EU, and the European Union NextGenerationEU/PRTR, supporting projects PID2020-112758RB-I00 (M.F.), PDC2021-121534-I00 (M.F.), TED2021-130544B-I00 (M.F.), PID2023-153370OB-I00 (M.F.), PID2019-106370RB-I00 (V.G.), and PID2022–136367OB-C31/C33 (F.J.P. and J.S.). We thank the Synchrotron Radiation Source at ALBA (Barcelona, Spain) for assisting with the BL13-XALOC beamline.Peer reviewedElsevierEuropean CommissionMinisterio de Ciencia, Innovación y Universidades (España)Agencia Estatal de Investigación (España)Muñoz Tafalla, Rubén [0000-0003-4380-4888]Cea Rama, Isabel [0000-0002-0929-2076]Cervantes, Fadia V. [0000-0001-8844-8392]González-Alfonso, José L. [0000-0002-3396-7985]Plou, Francisco J. [0000-0003-0831-893X]Polaina, Julio [0000-0001-9912-0640]Sanz Aparicio, Julia [0000-0002-6849-8621]Ferrer, Manuel [0000-0003-4962-4714]Guallar, Víctor [0000-0002-4580-1114]Talens Perales, David [0000-0002-8693-4239]Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]202520252025info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionapplication/pdfhttp://hdl.handle.net/10261/401526https://api.elsevier.com/content/abstract/scopus_id/105015144868reponame:DIGITAL.CSIC. 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