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

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dc.citation.titlePLoS ONE
dc.citation.volume11
dc.creatorFuentealba, Matías
dc.creatorMuñoz, Rodrigo
dc.creatorMaturana, Pablo
dc.creatorKrapp, Adriana R.
dc.creatorCabrera, Ricardo
dc.date.accessioned2024-06-04T19:54:49Z
dc.date.available2024-06-04T19:54:49Z
dc.date.issued2016-03-24
dc.description.abstractGlucose 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.
dc.description.filFil: Fuentealba, Matías. Universidad de Chile. Facultad de Ciencias. Laboratorio de Bioquímica y Biología Molecular; Chile.
dc.description.filFil: Muñoz, Rodrigo. Universidad de Chile. Facultad de Ciencias. Laboratorio de Bioquímica y Biología Molecular; Chile.
dc.description.filFil: Maturana, Pablo. Universidad de Chile. Facultad de Ciencias. Laboratorio de Bioquímica y Biología Molecular; Chile.
dc.description.filFil: Krapp, Adriana R. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.
dc.description.filFil: Cabrera, Ricardo. Universidad de Chile. Facultad de Ciencias. Laboratorio de Bioquímica y Biología Molecular; Chile.
dc.description.sponsorshipFondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT): 1121170
dc.description.sponsorshipProyectos Anillos CONICYT: ACT-1107
dc.description.versionpeerreviewed
dc.format.extent1-22
dc.identifier.issn1932-6203
dc.identifier.urihttps://hdl.handle.net/2133/27208
dc.language.isoen
dc.publisherPublic Library of Science (PLOS)
dc.relation.publisherversionhttps://doi.org/10.1371/journal.pone.0152403
dc.relation.publisherversionhttps://journals.plos.org/plosone/article?id=10.1371/journal.pone.0152403
dc.rightsopenAccess
dc.rights.holderFuentealba, Matías
dc.rights.holderMuñoz, Rodrigo
dc.rights.holderMaturana, Pablo
dc.rights.holderKrapp, Adriana R.
dc.rights.holderCabrera, Ricardo
dc.rights.holderUniversidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas
dc.rights.textAttribution 4.0 International
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/
dc.subjectEscherichia coli
dc.subjectGlucose-6-Phosphate Dehydrogenase
dc.subjectGlucosephosphate dehydrogenase
dc.subjectSensitivity and specificity
dc.subjectNAD
dc.subjectNADP
dc.subjectMolecular dynamics simulation
dc.subjectKinetics
dc.subjectPhylogeny
dc.titleDeterminants of cofactor specificity for the glucose-6-phosphate dehydrogenase from Escherichia coli: simulation, kinetics and evolutionary studies
dc.typearticulo
dc.type.collectionarticulo
dc.type.versionpublishedVersion

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