title: Determinants of Cofactor Specificity for the Glucose-6-Phosphate Dehydrogenase from Escherichia coli: Simulation, Kinetics and Evolutionary Studies.
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.
authors.affiliation: Laboratorio de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
authors.forename: Matias
authors.initials: M
authors.lastname: Fuentealba
authors.affiliation: Laboratorio de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Muñoz
authors.affiliation: Laboratorio de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
authors.forename: Pablo
authors.initials: P
authors.lastname: Maturana
authors.affiliation: Instituto de Biología Molecular y Celular de Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
authors.forename: Adriana
authors.initials: A
authors.lastname: Krapp
authors.affiliation: Laboratorio de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Cabrera
mesh_terms: D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D001499:Bayes Theorem; D001665:Binding Sites; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D019143:Evolution, Molecular; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry*; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009249:NADP / Q000737:chemistry; D010802:Phylogeny; D010957:Plasmids / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D009249:NADP; D005954:Glucosephosphate Dehydrogenase; D000409:Alanine