科研之友
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摘要
In most living organisms, isocitrate dehydrogenases (IDHs) convert isocitrate into alpha-ketoglutarate (alpha-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 (CIIDH, 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 mu M or 309.1 +/- 11.2 mu M 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 CIIDH is an NAD(+)-specific ancestral enzyme in the type II IDH subgroup. Gel filtration and analytical ultracentrifugation analyses revealed the homohexameric structure of CIIDH, 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. CIIDH was dependent on divalent cations, the most effective being Mn2+. The maximal activity of purified recombinant CIIDH was achieved at 35 degrees C and pH 7.5, and a heat inactivation experiment showed that a 20-min incubation at 33 degrees C caused a 50% loss of CIIDH 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.
