LAUSR

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 (Leu584/Asp595/Ser644), a typical NADP+-binding motif was also identified in Campylobacter corcagiensis IDH (CcoIDH, His582/Arg593/Arg638). Meanwhile, a third putative NAD+-binding motif was found in Campylobacter concisus IDH (CcIDH, Leu580/Leu591/Ala640). 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 Km value of CcIDH for NADP+ was over 49-fold higher than that for NAD+ and the catalytic efficiency (kcat/Km) of CcIDH is 115-fold greater for NAD+ than NADP+. Thus, CcIDH is indeed an NAD+-specific enzyme. However, the catalytic efficiency (kcat/Km = 0.886 μM-1 s-1) 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 (H580R591R640) showed a 51-fold higher Km value for NAD+ and 21-fold lower Km 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 H580R591R640 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.