Characterization of the nicotinamide adenine dinucleotides (NAD and NADP) binding sites of the monomeric isocitrate dehydrogenases from Campylobacter species.

Monomeric isocitrate dehydrogenases (IDHs) have once been proposed to be exclusively NADP-specific. Intriguingly, we recently have reported an NAD-specific monomeric IDH from Campylobacter sp. FOBRC14 (CaIDH). Moreover, bioinformatic analysis revealed at least three different coenzyme-binding motifs among Campylobacter IDHs. Besides the NAD-binding motif in CaIDH (Leu/Asp/Ser), a typical NADP-binding motif was also identified in Campylobacter corcagiensis IDH (CcoIDH, His/Arg/Arg). Meanwhile, a third putative NAD-binding motif was found in Campylobacter concisus IDH (CcIDH, Leu/Leu/Ala). In this study, CcIDH was overexpressed in Escherichia coli and purified to homogeneity. Gel filtration chromatography demonstrated that the recombinant CcIDH exists as a monomer in solution. Kinetic analysis showed that the K value of CcIDH for NADP was over 49-fold higher than that for NAD and the catalytic efficiency (k/K) of CcIDH is 115-fold greater for NAD than NADP. Thus, CcIDH is indeed an NAD-specific enzyme. However, the catalytic efficiency (k/K = 0.886 μM s) of CcIDH for NAD is much lower (<5%) when compared to that of the typical monomeric NADP-IDHs for NADP. Then, the three core NAD-binding sites were evaluated by site-directed mutagenesis. The mutant CcIDH (HRR) showed a 51-fold higher K value for NAD and 21-fold lower K value for NADP as compared to that of the wild type enzyme, respectively. The overall specificity of the mutant CcIDH was 12-fold greater for NADP than that for NAD. Thus, the coenzyme specificity of CcIDH was converted from NAD to NADP. Isocitrate dependence of enzyme kinetics showed that although the mutant HRR preferred NADP as its coenzyme, its catalytic efficiency for isocitrate reduced to half of that for the wild-type CcIDH as using NAD. The finding of both NAD and NADP-binding sites in monomeric IDHs from Campylobacter species will provide us a chance to explore the evolution of the coenzyme specificity in monomeric IDH subfamily.