Protein flexibility drives sugar rotation and high substrate promiscuity in a GDP-sugar 4-epimerase

UDP-galactose 4-epimerases (Gal4Es) catalyze the inversion of the 4-hydroxyl configuration of a sugar moiety from an NDP-sugar through a three-step process: oxidation, rotation and reduction. Despite extensive biochemical and structural studies, the role of protein dynamics on substrate specificity...

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Autores: Alvarez Quispe, Carlos Josue, Beerens, Koen, Thunnissen, Andy-Mark, Biarnés, Xevi, Planas, Antoni, Desmet, Tom
Tipo de recurso: artículo
Fecha de publicación:2025
País:España
Institución:Universitat Ramon Llull (URL)
Repositorio:DAU Arxiu Digital de la Universitat Ramon Llull
OAI Identifier:oai:dau.url.edu:20.500.14342/5350
Acceso en línea:http://hdl.handle.net/20.500.14342/5350
https://doi.org/10.1016/j.csbj.2025.05.037
Access Level:acceso abierto
Palabra clave:Carbohydrate epimerases (CEP1)
GDP-sugar 4-epimerase
UDP-galactose 4-epimerase
Nucleotide-sugars
Heptagonal box model
Sugar ring rotation
Molecular dynamics simulations
L-sugars
Dinàmica molecular
577
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oai_identifier_str oai:dau.url.edu:20.500.14342/5350
network_acronym_str ES
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repository_id_str
spelling Protein flexibility drives sugar rotation and high substrate promiscuity in a GDP-sugar 4-epimeraseAlvarez Quispe, Carlos JosueBeerens, KoenThunnissen, Andy-MarkBiarnés, XeviPlanas, AntoniDesmet, TomCarbohydrate epimerases (CEP1)GDP-sugar 4-epimeraseUDP-galactose 4-epimeraseNucleotide-sugarsHeptagonal box modelSugar ring rotationMolecular dynamics simulationsL-sugarsDinàmica molecular577UDP-galactose 4-epimerases (Gal4Es) catalyze the inversion of the 4-hydroxyl configuration of a sugar moiety from an NDP-sugar through a three-step process: oxidation, rotation and reduction. Despite extensive biochemical and structural studies, the role of protein dynamics on substrate specificity remains poorly understood. The recently identified subgroup of GDP-sugar 4-epimerases, notable for its exceptional substrate promiscuity, provides an intriguing model to investigate the role of dynamics in the Gal4E catalytic mechanism and the unique promiscuity of the subgroup. In this study, we used a multidisciplinary approach to examine the dynamic-function relationships in the Pyrococcus horikoshii representative (PhGal4E_1). First, we determined several crystal structures (WT: 1.9–2.4 Å and Y145F: 3.1 Å), providing structural insights of the PhGal4E_1 structure bound to GDP-L-fucose in a catalytic conformation. To further explore the enzyme’s promiscuity, in silico docking studies were conducted with three substrates, namely GDP-L-Fuc, GDP-Glc and UDP-Glc. Molecular dynamics simulations identified a dynamic hydrogen bond network surrounding the sugar moiety and phosphate groups, revealing four key residues: P80, H182, R83 and N174. These residues interact with either the substrate’s sugar moiety (H182 and P80 with C2-OH and C3-OH, resp.) or diphosphate backbone (N174 and R83 with β-/α- and α-phosphate, resp.), which facilitates sugar ring positioning. Protein flexibility then initiates disruption of the hydrogen bonds enabling the required rotation of the intermediate. Site directed mutagenesis of these residues was performed to disrupt the interaction network followed by enzyme activity assays on the three substrates, validating their critical role in the epimerization reaction. These results highlight the pivotal role of protein flexibility in PhGal4E_1 promiscuity and establish a framework for dynamic studies across other Gal4E representatives.info:eu-repo/semantics/publishedVersionElsevierUniversitat Ramon Llull. IQS202520252025info:eu-repo/semantics/articlep.11application/pdfhttp://hdl.handle.net/20.500.14342/5350https://doi.org/10.1016/j.csbj.2025.05.037reponame:DAU Arxiu Digital de la Universitat Ramon Llullinstname:Universitat Ramon Llull (URL)InglésResearch Network of Computational and Structural Biotechnology 2025, 27, 2375-2385info:eu-repo/grantAgreement/MCI/PN I+D/PID2022-138252OB-I00© L'autor/aAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccessoai:dau.url.edu:20.500.14342/53502026-06-21T06:40:37Z
dc.title.none.fl_str_mv Protein flexibility drives sugar rotation and high substrate promiscuity in a GDP-sugar 4-epimerase
title Protein flexibility drives sugar rotation and high substrate promiscuity in a GDP-sugar 4-epimerase
spellingShingle Protein flexibility drives sugar rotation and high substrate promiscuity in a GDP-sugar 4-epimerase
Alvarez Quispe, Carlos Josue
Carbohydrate epimerases (CEP1)
GDP-sugar 4-epimerase
UDP-galactose 4-epimerase
Nucleotide-sugars
Heptagonal box model
Sugar ring rotation
Molecular dynamics simulations
L-sugars
Dinàmica molecular
577
title_short Protein flexibility drives sugar rotation and high substrate promiscuity in a GDP-sugar 4-epimerase
title_full Protein flexibility drives sugar rotation and high substrate promiscuity in a GDP-sugar 4-epimerase
title_fullStr Protein flexibility drives sugar rotation and high substrate promiscuity in a GDP-sugar 4-epimerase
title_full_unstemmed Protein flexibility drives sugar rotation and high substrate promiscuity in a GDP-sugar 4-epimerase
title_sort Protein flexibility drives sugar rotation and high substrate promiscuity in a GDP-sugar 4-epimerase
dc.creator.none.fl_str_mv Alvarez Quispe, Carlos Josue
Beerens, Koen
Thunnissen, Andy-Mark
Biarnés, Xevi
Planas, Antoni
Desmet, Tom
author Alvarez Quispe, Carlos Josue
author_facet Alvarez Quispe, Carlos Josue
Beerens, Koen
Thunnissen, Andy-Mark
Biarnés, Xevi
Planas, Antoni
Desmet, Tom
author_role author
author2 Beerens, Koen
Thunnissen, Andy-Mark
Biarnés, Xevi
Planas, Antoni
Desmet, Tom
author2_role author
author
author
author
author
dc.contributor.none.fl_str_mv Universitat Ramon Llull. IQS
dc.subject.none.fl_str_mv Carbohydrate epimerases (CEP1)
GDP-sugar 4-epimerase
UDP-galactose 4-epimerase
Nucleotide-sugars
Heptagonal box model
Sugar ring rotation
Molecular dynamics simulations
L-sugars
Dinàmica molecular
577
topic Carbohydrate epimerases (CEP1)
GDP-sugar 4-epimerase
UDP-galactose 4-epimerase
Nucleotide-sugars
Heptagonal box model
Sugar ring rotation
Molecular dynamics simulations
L-sugars
Dinàmica molecular
577
description UDP-galactose 4-epimerases (Gal4Es) catalyze the inversion of the 4-hydroxyl configuration of a sugar moiety from an NDP-sugar through a three-step process: oxidation, rotation and reduction. Despite extensive biochemical and structural studies, the role of protein dynamics on substrate specificity remains poorly understood. The recently identified subgroup of GDP-sugar 4-epimerases, notable for its exceptional substrate promiscuity, provides an intriguing model to investigate the role of dynamics in the Gal4E catalytic mechanism and the unique promiscuity of the subgroup. In this study, we used a multidisciplinary approach to examine the dynamic-function relationships in the Pyrococcus horikoshii representative (PhGal4E_1). First, we determined several crystal structures (WT: 1.9–2.4 Å and Y145F: 3.1 Å), providing structural insights of the PhGal4E_1 structure bound to GDP-L-fucose in a catalytic conformation. To further explore the enzyme’s promiscuity, in silico docking studies were conducted with three substrates, namely GDP-L-Fuc, GDP-Glc and UDP-Glc. Molecular dynamics simulations identified a dynamic hydrogen bond network surrounding the sugar moiety and phosphate groups, revealing four key residues: P80, H182, R83 and N174. These residues interact with either the substrate’s sugar moiety (H182 and P80 with C2-OH and C3-OH, resp.) or diphosphate backbone (N174 and R83 with β-/α- and α-phosphate, resp.), which facilitates sugar ring positioning. Protein flexibility then initiates disruption of the hydrogen bonds enabling the required rotation of the intermediate. Site directed mutagenesis of these residues was performed to disrupt the interaction network followed by enzyme activity assays on the three substrates, validating their critical role in the epimerization reaction. These results highlight the pivotal role of protein flexibility in PhGal4E_1 promiscuity and establish a framework for dynamic studies across other Gal4E representatives.
publishDate 2025
dc.date.none.fl_str_mv 2025
2025
2025
dc.type.none.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv http://hdl.handle.net/20.500.14342/5350
https://doi.org/10.1016/j.csbj.2025.05.037
url http://hdl.handle.net/20.500.14342/5350
https://doi.org/10.1016/j.csbj.2025.05.037
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv Research Network of Computational and Structural Biotechnology 2025, 27, 2375-2385
info:eu-repo/grantAgreement/MCI/PN I+D/PID2022-138252OB-I00
dc.rights.none.fl_str_mv © L'autor/a
Attribution-NonCommercial-NoDerivatives 4.0 International
http://creativecommons.org/licenses/by-nc-nd/4.0/
info:eu-repo/semantics/openAccess
rights_invalid_str_mv © L'autor/a
Attribution-NonCommercial-NoDerivatives 4.0 International
http://creativecommons.org/licenses/by-nc-nd/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv p.11
application/pdf
dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
dc.source.none.fl_str_mv reponame:DAU Arxiu Digital de la Universitat Ramon Llull
instname:Universitat Ramon Llull (URL)
instname_str Universitat Ramon Llull (URL)
reponame_str DAU Arxiu Digital de la Universitat Ramon Llull
collection DAU Arxiu Digital de la Universitat Ramon Llull
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,812455

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