Determinants of cofactor specificity for the glucose-6-phosphate dehydrogenase from Escherichia coli: simulation, kinetics and evolutionary studies
| dc.citation.title | PLoS ONE | |
| dc.citation.volume | 11 | |
| dc.creator | Fuentealba, Matías | |
| dc.creator | Muñoz, Rodrigo | |
| dc.creator | Maturana, Pablo | |
| dc.creator | Krapp, Adriana R. | |
| dc.creator | Cabrera, Ricardo | |
| dc.date.accessioned | 2024-06-04T19:54:49Z | |
| dc.date.available | 2024-06-04T19:54:49Z | |
| dc.date.issued | 2016-03-24 | |
| dc.description.abstract | 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. | |
| dc.description.fil | Fil: Fuentealba, Matías. Universidad de Chile. Facultad de Ciencias. Laboratorio de Bioquímica y Biología Molecular; Chile. | |
| dc.description.fil | Fil: Muñoz, Rodrigo. Universidad de Chile. Facultad de Ciencias. Laboratorio de Bioquímica y Biología Molecular; Chile. | |
| dc.description.fil | Fil: Maturana, Pablo. Universidad de Chile. Facultad de Ciencias. Laboratorio de Bioquímica y Biología Molecular; Chile. | |
| dc.description.fil | Fil: 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.fil | Fil: Cabrera, Ricardo. Universidad de Chile. Facultad de Ciencias. Laboratorio de Bioquímica y Biología Molecular; Chile. | |
| dc.description.sponsorship | Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT): 1121170 | |
| dc.description.sponsorship | Proyectos Anillos CONICYT: ACT-1107 | |
| dc.description.version | peerreviewed | |
| dc.format.extent | 1-22 | |
| dc.identifier.issn | 1932-6203 | |
| dc.identifier.uri | https://hdl.handle.net/2133/27208 | |
| dc.language.iso | en | |
| dc.publisher | Public Library of Science (PLOS) | |
| dc.relation.publisherversion | https://doi.org/10.1371/journal.pone.0152403 | |
| dc.relation.publisherversion | https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0152403 | |
| dc.rights | openAccess | |
| dc.rights.holder | Fuentealba, Matías | |
| dc.rights.holder | Muñoz, Rodrigo | |
| dc.rights.holder | Maturana, Pablo | |
| dc.rights.holder | Krapp, Adriana R. | |
| dc.rights.holder | Cabrera, Ricardo | |
| dc.rights.holder | Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas | |
| dc.rights.text | Attribution 4.0 International | |
| dc.rights.uri | http://creativecommons.org/licenses/by/4.0/ | |
| dc.subject | Escherichia coli | |
| dc.subject | Glucose-6-Phosphate Dehydrogenase | |
| dc.subject | Glucosephosphate dehydrogenase | |
| dc.subject | Sensitivity and specificity | |
| dc.subject | NAD | |
| dc.subject | NADP | |
| dc.subject | Molecular dynamics simulation | |
| dc.subject | Kinetics | |
| dc.subject | Phylogeny | |
| dc.title | Determinants of cofactor specificity for the glucose-6-phosphate dehydrogenase from Escherichia coli: simulation, kinetics and evolutionary studies | |
| dc.type | articulo | |
| dc.type.collection | articulo | |
| dc.type.version | publishedVersion |