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
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/52401
Ver los metadatos del registro completo
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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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13.070432 |