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

Abstract

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 K_m value of CcIDH for NADP⁺ was over 49-fold higher than that for NAD⁺ and the catalytic efficiency (k_cat/K_m) of CcIDH is 115-fold greater for NAD⁺ than NADP⁺. Thus, CcIDH is indeed an NAD⁺-specific enzyme. However, the catalytic efficiency (k_cat/K_m = 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 K_m value for NAD⁺ and 21-fold lower K_m 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.

Keywords: Campylobacter species; Coenzyme binding site; Evolution; Monomeric isocitrate dehydrogenase; Site-directed mutagenesis.