A Novel Type II NAD+-Specific Isocitrate Dehydrogenase from the Marine Bacterium Congregibacter litoralis KT71

Abstract

In most living organisms, isocitrate dehydrogenases (IDHs) convert isocitrate into ɑ-ketoglutarate (ɑ-KG). Phylogenetic analyses divide the IDH protein family into two subgroups: types I and II. Based on cofactor usage, IDHs are either NAD+-specific (NAD-IDH) or NADP+-specific (NADP-IDH); NADP-IDH evolved from NAD-IDH. Type I IDHs include NAD-IDHs and NADP-IDHs; however, no type II NAD-IDHs have been reported to date. This study reports a novel type II NAD-IDH from the marine bacterium Congregibacter litoralis KT71 (ClIDH, GenBank accession no. EAQ96042). His-tagged recombinant ClIDH was produced in Escherichia coli and purified; the recombinant enzyme was NAD+-specific and showed no detectable activity with NADP+. The Km values of the enzyme for NAD+ were 262.6±7.4 μM or 309.1±11.2 μM with Mg2+ or Mn2+ as the divalent cation, respectively. The coenzyme specificity of a ClIDH Asp487Arg/Leu488His mutant was altered, and the preference of the mutant for NADP+ was approximately 24-fold higher than that for NAD+, suggesting that ClIDH is an NAD+-specific ancestral enzyme in the type II IDH subgroup. Gel filtration and analytical ultracentrifugation analyses revealed the homohexameric structure of ClIDH, which is the first IDH hexamer discovered thus far. A 163-amino acid segment of CIIDH is essential to maintain its polymerization structure and activity, as a truncated version lacking this region forms a non-functional monomer. ClIDH was dependent on divalent cations, the most effective being Mn2+. The maximal activity of purified recombinant ClIDH was achieved at 35°C and pH 7.5, and a heat inactivation experiment showed that a 20-min incubation at 33°C caused a 50% loss of ClIDH activity. The discovery of a NAD+-specific, type II IDH fills a gap in the current classification of IDHs, and sheds light on the evolution of type II IDHs.

Fig 1. Evolutionary relationships between 151 IDHs from different organisms.

Green branches represent NADP-IDHs, and pink branches represent NAD-IDHs. The evolutionary history was inferred using the neighbor-joining method [35]. The bootstrap consensus tree inferred from 500 replicates represents the evolutionary history of the analyzed taxa [36]. Branches corresponding to partitions that are reproduced in less than 50% of the bootstrap replicates are collapsed. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches [36]. The tree is drawn to scale, with branch lengths presented in the same units as the evolutionary distances that were used to infer-he phylogenetic tree. The evolutionary distances were computed using the Poisson correction method [37] and are shown in units of ‘number of amino acid substitutions per site’. All positions containing gaps and missing data were eliminated from the dataset (complete deletion option). A total of 255 positions were in the final dataset. Phylogenetic analyses were conducted using MEGA4 [15].

Fig 2. Structure-based protein sequence alignment of ClIDH with three typical type II homodimeric IDHs.

The three typical type II homodimeric IDHs were from Homo sapiens (human) (HsIDH, GenBank accession no. NP_005887.2), Saccharomyces cerevisiae (yeast) (ScIDH, GenBank accession no. P21954) and Mycobacterium tuberculosis (MtIDH, GenBank accession no. WP_003904258.1). The high-resolution structure of MtIDH (PDB ID, 4HCX) was downloaded from the PDB database. The ClIDH homology model was generated using the SWISS-MODEL server. Invariant residues are highlighted with shaded blue boxes, and conserved residues are highlighted with open blue boxes. The conserved residues that are involved in cofactor binding (★) and substrate binding (▲) are indicated. The clasp region is represented by (*), and the small domain is represented by (♣). The figure was generated using ESPript 2.2 [14].

Fig 3. Purification of recombinant ClIDH.

(a) Protein purity was assessed via 12% SDS-PAGE. M, protein marker; lane 1, crude extracts of cells harboring pET-28b(+) after induction with IPTG; lane 2, crude extracts of cells harboring the recombinant plasmid after induction with IPTG; lane 3, purified protein. (b) Western blot analysis using the anti-6×His antibody as a probe. Lane 1, negative control, crude extracts of cells harboring pET-28b(+) with IPTG induction; lane 2, purified protein. (c) Molecular mass determination via gel filtration chromatography. The V_e of recombinant ClIDH was 10.7 mL.

Fig 4. Histogram plot showing the sedimentation coefficient distribution of ClIDH at 20°C.

Fig 5. Molecular mass determination of truncated ClIDH via gel filtration chromatography.

The V_e of the truncated ClIDH is 14.77 mL.

Fig 6. Effects of pH and temperature on the activity of purified recombinant ClIDH.

(a) Effects of temperature, from 20°C to 45°C, on enzyme activity in the presence of Mg²⁺ (●) or Mn²⁺ (○). (b) Effects of pH 7.0 to 9.0 on enzyme activity in the presence of Mg²⁺ (●) or Mn²⁺ (○). (c) Heat inactivation profiles of recombinant ClIDH. The activity was measured after 20 min incubation of the enzyme at the temperatures from 25°C to 38°C in the presence of Mg²⁺ (●) or Mn²⁺ (○).