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.

Cloning, Expression, and Characterization of a Psychrophilic Glucose 6-Phosphate Dehydrogenase from Sphingomonas sp. PAMC 26621

“…The kinetic data reveal a significant difference in the K m values for G6P in NADP + -linked and NAD + -linked reactions; the ( K mG6P NAD+ )/( K m G6P NADP+ ) ratio of SpG6PD1 was 4.7, which is higher than that obtained for Streptomyces aureofaciens [37], Leuconostoc mesenteroides [16], and Methylomonas M15 [38], but lower than Thermoanaerobacter tengcongensis [6]. These strains are classified as dual cofactor-specific or NADP + -preferring G6PD enzymes [23]. Thus, SpG6PD1 also belongs to the enzyme category, which utilizes both NADP + and NAD + as cofactors.…”

“…SpG6PD1 ( Sphingomonas sp. PAMC 26621), SeG6PD ( Sphingomonas elodea [20]), PfG6PD ( Pseudomonas fluorescens [21]), LmG6PD ( Leuconostoc mesenteroides [5]), HmG6PD (Human [22]), EcG6PD ( Escherichia coli [23]), CdG6PD ( Camelus dromedarius [24]), HvG6PD ( Hordeum vulgare [25]), AqG6PD ( Aquifex aeolicus VF5 [7]), TtG6PD ( Thermoanaerobacter tengcongensis [6]), TmG6PD ( Thermotoga maritima [26]). Catalytic residues Asp174, His175, and His237 (turquoise color), the substrate-binding motifs (yellow color), cofactor-binding pockets (green color).…”

Cofactor specificity of glucose-6-phosphate dehydrogenase isozymes in Pseudomonas putida reveals a general principle underlying glycolytic strategies in bacteria

“…Building on this rationale, this amino acid residue (arginine) was found to be determinant for the usage of NADP + by the G6PDHs from several organisms, including E. coli (53) and the lactic acid bacterium L. mesenteroides (54, 55). Furthermore, the G6PDH isozymes of Komagataeibacter hansenii , Burkholderia cepacia and Pseudomonas fluorescence (biotype E), which are the most NAD + -specific G6DPH characterized so far, lack this specific arginine residue (16, 18, 44, 53, 56). Thus, we reasoned that if a given G6PDH contains an arginine residue at the position, predicted to interact with the 2′-phosphate moiety of NADP + , it can be considered either NADP + or dual cofactor-specific, whereas other amino acids in this position would lower the NADP + -affinity of the enzyme.…”

“…G6PDHs from several organisms, including E. coli (53) and the lactic acid bacterium L. mesenteroides (54,55). Furthermore, the G6PDH isozymes of Komagataeibacter hansenii, Burkholderia cepacia and Pseudomonas fluorescence (biotype E), which are the most NAD + -specific G6DPH characterized so far, lack this specific arginine residue (16, 18, 44, 53, 56).…”

Sterol Regulatory Element-Binding Protein (Sre1) Promotes the Synthesis of Carotenoids and Sterols in Xanthophyllomyces dendrorhous

“…EMSAs were performed using the deduced DNA-binding bHLH motif protein of the X. dendrorhous Sre1 (residues 349 to 477) fused to a His-tag, which was synthesized in E. coli BL21 (DE3) using plasmid pET-TEV (Fuentealba et al, 2016). Gene expression was induced with 1 mM IPTG using exponential growth cultures grown in LB medium at 22°C containing the corresponding antibiotics.…”