A Novel Type II NAD+-Specific Isocitrate Dehydrogenase from the Marine Bacterium Congregibacter litoralis KT71.
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.
Institute of Molecular Biology and Biotechnology, Anhui Normal University, No. 1 Beijing East Road, Wuhu, 241000, Anhui, China; Anhui Province Key Laboratory of Active Biological Macro-molecules, Wannan Medical College, No. 22 Wenchang West Road, Wuhu, 241002, Anhui, China.
Ming-Cai
MC
Wu
Institute of Molecular Biology and Biotechnology, Anhui Normal University, No. 1 Beijing East Road, Wuhu, 241000, Anhui, China.
Chang-Qing
CQ
Tian
Institute of Molecular Biology and Biotechnology, Anhui Normal University, No. 1 Beijing East Road, Wuhu, 241000, Anhui, China.
Hong-Mei
HM
Cheng
Institute of Molecular Biology and Biotechnology, Anhui Normal University, No. 1 Beijing East Road, Wuhu, 241000, Anhui, China; Anhui Province Key Laboratory of Active Biological Macro-molecules, Wannan Medical College, No. 22 Wenchang West Road, Wuhu, 241002, Anhui, China.
Lei
L
Xu
Institute of Molecular Biology and Biotechnology, Anhui Normal University, No. 1 Beijing East Road, Wuhu, 241000, Anhui, China.
Peng
P
Wang
Institute of Molecular Biology and Biotechnology, Anhui Normal University, No. 1 Beijing East Road, Wuhu, 241000, Anhui, China.
Guo-Ping
GP
Zhu
D000595:Amino Acid Sequence; D004789:Enzyme Activation; D020563:Gammaproteobacteria / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D007521:Isocitrate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009243:NAD / Q000378:metabolism; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D011994:Recombinant Proteins; D009243:NAD; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+)