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.
Highlights
Substrate recognition depends on the interactions between the chemical groups of the ligand and the amino acid residues at the active site of the enzyme
We found that the use of NADP+ as the preferred cofactor seems to be a common feature in part of the Glucose 6-Phosphate Dehydrogenases (G6PDHs) family and it is associated with the conservation of R50
In order to characterize differences between the interactions made by the nicotinamide dinucleotides with residues in the binding pocket of EcG6PDH, we analyzed molecular dynamics simulations of complexes with these ligands
Summary
Substrate recognition depends on the interactions between the chemical groups of the ligand and the amino acid residues at the active site of the enzyme. NAD+ and NADP+ are alternative cofactors whose differences in binding energy, determined by the presence of the 2’-phosphate, result in most of these enzymes being highly selective towards one of the cofactors. At their active sites, determinants of cofactor specificity often occur within conserved sequence motifs. Five levels of cofactor specificity in G6PDHs were proposed, including NADP+- and NAD+-specific, NADP+- and NAD+-preferring, and dual enzymes. This initial classification was based on data of varying degrees of completeness and the distinctions were somewhat arbitrary. In the case of G6PDHs the previous assessments have not been explicitly based on this consideration
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