Determinants of cofactor specificity for the glucose-6-phosphate dehydrogenase from Escherichia coli: Simulation, kinetics and evolutionary studies

Autores
Fuentealba, Matias; Muñoz, Rodrigo; Maturana, Pablo; Krapp, Adriana del Rosario; Cabrera, Ricardo
Año de publicación
2016
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Glucose 6-Phosphate Dehydrogenases (G6PDHs) from different sources show varying specificities towards NAD+ and NADP+ as cofactors. However, it is not known to what extent structural determinants of cofactor preference are conserved in the G6PDH family. In this work, molecular simulations, kinetic characterization of site-directed mutants and phylogenetic analyses were used to study the structural basis for the strong preference towards NADP+ shown by the G6PDH from Escherichia coli. Molecular Dynamics trajectories of homology models showed a highly favorable binding energy for residues K18 and R50 when interacting with the 2'-phosphate of NADP+, but the same residues formed no observable interactions in the case of NAD+. Alanine mutants of both residues were kinetically characterized and analyzed with respect to the binding energy of the transition state, according to the kcat/KM value determined for each cofactor. Whereas both residues contribute to the binding energy of NADP+, only R50 makes a contribution (about -1 kcal/mol) to NAD+ binding. In the absence of both positive charges the enzyme was unable to discriminate NADP+ from NAD+. Although kinetic data is sparse, the observed distribution of cofactor preferences within the phylogenetic tree is sufficient to rule out the possibility that the known NADP+-specific G6PDHs form a monophyletic group. While the β1-α1 loop shows no strict conservation of K18, (rather, S and T seem to be more frequent), in the case of the β2-α2 loop, different degrees of conservation are observed for R50. Noteworthy is the fact that a K18T mutant is indistinguishable from K18A in terms of cofactor preference. We conclude that the structural determinants for the strict discrimination against NAD+ in the case of the NADP+-specific enzymes have evolved independently through different means during the evolution of the G6PDH family. We further suggest that other regions in the cofactor binding pocket, besides the β1-α1 and β2-α2 loops, play a role in determining cofactor preference.
Fil: Fuentealba, Matias. Universidad de Chile; Chile
Fil: Muñoz, Rodrigo. Universidad de Chile; Chile
Fil: Maturana, Pablo. Universidad de Chile; Chile
Fil: Krapp, Adriana del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Cabrera, Ricardo. Universidad de Chile; Chile
Materia
G6PDH
SPECIFICITY
COFACTOR
NADP
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/52401

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repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Determinants of cofactor specificity for the glucose-6-phosphate dehydrogenase from Escherichia coli: Simulation, kinetics and evolutionary studiesFuentealba, MatiasMuñoz, RodrigoMaturana, PabloKrapp, Adriana del RosarioCabrera, RicardoG6PDHSPECIFICITYCOFACTORNADPhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Glucose 6-Phosphate Dehydrogenases (G6PDHs) from different sources show varying specificities towards NAD+ and NADP+ as cofactors. However, it is not known to what extent structural determinants of cofactor preference are conserved in the G6PDH family. In this work, molecular simulations, kinetic characterization of site-directed mutants and phylogenetic analyses were used to study the structural basis for the strong preference towards NADP+ shown by the G6PDH from Escherichia coli. Molecular Dynamics trajectories of homology models showed a highly favorable binding energy for residues K18 and R50 when interacting with the 2'-phosphate of NADP+, but the same residues formed no observable interactions in the case of NAD+. Alanine mutants of both residues were kinetically characterized and analyzed with respect to the binding energy of the transition state, according to the kcat/KM value determined for each cofactor. Whereas both residues contribute to the binding energy of NADP+, only R50 makes a contribution (about -1 kcal/mol) to NAD+ binding. In the absence of both positive charges the enzyme was unable to discriminate NADP+ from NAD+. Although kinetic data is sparse, the observed distribution of cofactor preferences within the phylogenetic tree is sufficient to rule out the possibility that the known NADP+-specific G6PDHs form a monophyletic group. While the β1-α1 loop shows no strict conservation of K18, (rather, S and T seem to be more frequent), in the case of the β2-α2 loop, different degrees of conservation are observed for R50. Noteworthy is the fact that a K18T mutant is indistinguishable from K18A in terms of cofactor preference. We conclude that the structural determinants for the strict discrimination against NAD+ in the case of the NADP+-specific enzymes have evolved independently through different means during the evolution of the G6PDH family. We further suggest that other regions in the cofactor binding pocket, besides the β1-α1 and β2-α2 loops, play a role in determining cofactor preference.Fil: Fuentealba, Matias. Universidad de Chile; ChileFil: Muñoz, Rodrigo. Universidad de Chile; ChileFil: Maturana, Pablo. Universidad de Chile; ChileFil: Krapp, Adriana del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Cabrera, Ricardo. Universidad de Chile; ChilePublic Library of Science2016-03info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/52401Fuentealba, Matias; Muñoz, Rodrigo; Maturana, Pablo; Krapp, Adriana del Rosario; Cabrera, Ricardo; Determinants of cofactor specificity for the glucose-6-phosphate dehydrogenase from Escherichia coli: Simulation, kinetics and evolutionary studies; Public Library of Science; Plos One; 11; 3; 3-2016; 1-22; e01524031932-6203CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1371/journal.pone.0152403info:eu-repo/semantics/altIdentifier/url/http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0152403info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:21:37Zoai:ri.conicet.gov.ar:11336/52401instacron: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:34982025-09-29 10:21:37.548CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Determinants of cofactor specificity for the glucose-6-phosphate dehydrogenase from Escherichia coli: Simulation, kinetics and evolutionary studies
title Determinants of cofactor specificity for the glucose-6-phosphate dehydrogenase from Escherichia coli: Simulation, kinetics and evolutionary studies
spellingShingle Determinants of cofactor specificity for the glucose-6-phosphate dehydrogenase from Escherichia coli: Simulation, kinetics and evolutionary studies
Fuentealba, Matias
G6PDH
SPECIFICITY
COFACTOR
NADP
title_short Determinants of cofactor specificity for the glucose-6-phosphate dehydrogenase from Escherichia coli: Simulation, kinetics and evolutionary studies
title_full Determinants of cofactor specificity for the glucose-6-phosphate dehydrogenase from Escherichia coli: Simulation, kinetics and evolutionary studies
title_fullStr Determinants of cofactor specificity for the glucose-6-phosphate dehydrogenase from Escherichia coli: Simulation, kinetics and evolutionary studies
title_full_unstemmed Determinants of cofactor specificity for the glucose-6-phosphate dehydrogenase from Escherichia coli: Simulation, kinetics and evolutionary studies
title_sort Determinants of cofactor specificity for the glucose-6-phosphate dehydrogenase from Escherichia coli: Simulation, kinetics and evolutionary studies
dc.creator.none.fl_str_mv Fuentealba, Matias
Muñoz, Rodrigo
Maturana, Pablo
Krapp, Adriana del Rosario
Cabrera, Ricardo
author Fuentealba, Matias
author_facet Fuentealba, Matias
Muñoz, Rodrigo
Maturana, Pablo
Krapp, Adriana del Rosario
Cabrera, Ricardo
author_role author
author2 Muñoz, Rodrigo
Maturana, Pablo
Krapp, Adriana del Rosario
Cabrera, Ricardo
author2_role author
author
author
author
dc.subject.none.fl_str_mv G6PDH
SPECIFICITY
COFACTOR
NADP
topic G6PDH
SPECIFICITY
COFACTOR
NADP
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Glucose 6-Phosphate Dehydrogenases (G6PDHs) from different sources show varying specificities towards NAD+ and NADP+ as cofactors. However, it is not known to what extent structural determinants of cofactor preference are conserved in the G6PDH family. In this work, molecular simulations, kinetic characterization of site-directed mutants and phylogenetic analyses were used to study the structural basis for the strong preference towards NADP+ shown by the G6PDH from Escherichia coli. Molecular Dynamics trajectories of homology models showed a highly favorable binding energy for residues K18 and R50 when interacting with the 2'-phosphate of NADP+, but the same residues formed no observable interactions in the case of NAD+. Alanine mutants of both residues were kinetically characterized and analyzed with respect to the binding energy of the transition state, according to the kcat/KM value determined for each cofactor. Whereas both residues contribute to the binding energy of NADP+, only R50 makes a contribution (about -1 kcal/mol) to NAD+ binding. In the absence of both positive charges the enzyme was unable to discriminate NADP+ from NAD+. Although kinetic data is sparse, the observed distribution of cofactor preferences within the phylogenetic tree is sufficient to rule out the possibility that the known NADP+-specific G6PDHs form a monophyletic group. While the β1-α1 loop shows no strict conservation of K18, (rather, S and T seem to be more frequent), in the case of the β2-α2 loop, different degrees of conservation are observed for R50. Noteworthy is the fact that a K18T mutant is indistinguishable from K18A in terms of cofactor preference. We conclude that the structural determinants for the strict discrimination against NAD+ in the case of the NADP+-specific enzymes have evolved independently through different means during the evolution of the G6PDH family. We further suggest that other regions in the cofactor binding pocket, besides the β1-α1 and β2-α2 loops, play a role in determining cofactor preference.
Fil: Fuentealba, Matias. Universidad de Chile; Chile
Fil: Muñoz, Rodrigo. Universidad de Chile; Chile
Fil: Maturana, Pablo. Universidad de Chile; Chile
Fil: Krapp, Adriana del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Cabrera, Ricardo. Universidad de Chile; Chile
description Glucose 6-Phosphate Dehydrogenases (G6PDHs) from different sources show varying specificities towards NAD+ and NADP+ as cofactors. However, it is not known to what extent structural determinants of cofactor preference are conserved in the G6PDH family. In this work, molecular simulations, kinetic characterization of site-directed mutants and phylogenetic analyses were used to study the structural basis for the strong preference towards NADP+ shown by the G6PDH from Escherichia coli. Molecular Dynamics trajectories of homology models showed a highly favorable binding energy for residues K18 and R50 when interacting with the 2'-phosphate of NADP+, but the same residues formed no observable interactions in the case of NAD+. Alanine mutants of both residues were kinetically characterized and analyzed with respect to the binding energy of the transition state, according to the kcat/KM value determined for each cofactor. Whereas both residues contribute to the binding energy of NADP+, only R50 makes a contribution (about -1 kcal/mol) to NAD+ binding. In the absence of both positive charges the enzyme was unable to discriminate NADP+ from NAD+. Although kinetic data is sparse, the observed distribution of cofactor preferences within the phylogenetic tree is sufficient to rule out the possibility that the known NADP+-specific G6PDHs form a monophyletic group. While the β1-α1 loop shows no strict conservation of K18, (rather, S and T seem to be more frequent), in the case of the β2-α2 loop, different degrees of conservation are observed for R50. Noteworthy is the fact that a K18T mutant is indistinguishable from K18A in terms of cofactor preference. We conclude that the structural determinants for the strict discrimination against NAD+ in the case of the NADP+-specific enzymes have evolved independently through different means during the evolution of the G6PDH family. We further suggest that other regions in the cofactor binding pocket, besides the β1-α1 and β2-α2 loops, play a role in determining cofactor preference.
publishDate 2016
dc.date.none.fl_str_mv 2016-03
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/11336/52401
Fuentealba, Matias; Muñoz, Rodrigo; Maturana, Pablo; Krapp, Adriana del Rosario; Cabrera, Ricardo; Determinants of cofactor specificity for the glucose-6-phosphate dehydrogenase from Escherichia coli: Simulation, kinetics and evolutionary studies; Public Library of Science; Plos One; 11; 3; 3-2016; 1-22; e0152403
1932-6203
CONICET Digital
CONICET
url http://hdl.handle.net/11336/52401
identifier_str_mv Fuentealba, Matias; Muñoz, Rodrigo; Maturana, Pablo; Krapp, Adriana del Rosario; Cabrera, Ricardo; Determinants of cofactor specificity for the glucose-6-phosphate dehydrogenase from Escherichia coli: Simulation, kinetics and evolutionary studies; Public Library of Science; Plos One; 11; 3; 3-2016; 1-22; e0152403
1932-6203
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1371/journal.pone.0152403
info:eu-repo/semantics/altIdentifier/url/http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0152403
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Public Library of Science
publisher.none.fl_str_mv Public Library of Science
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
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