Characterization of isocitrate dehydrogenase from the green sulfur bacterium Chlorobium limicola. A carbon dioxide-fixing enzyme in the reductive tricarboxylic acid cycle.

Isocitrate dehydrogenase (IDH) catalyzes the reversible conversion between isocitrate and 2-oxoglutarate accompanied by decarboxylation/carboxylation and oxidoreduction of NAD(P)+ cofactor. While this enzyme has been well studied as a catabolic enzyme in the tricarboxylic acid (TCA) cycle, here we have characterized NADP-dependent IDH from Chlorobium limicola, a green sulfur bacterium that fixes CO2 through the reductive tricarboxylic acid (RTCA) cycle, focusing on the CO2-fixation ability of the enzyme. The gene encoding Cl-IDH consisted of 2226 bp, corresponding to a polypeptide of 742 amino acid residues. The primary structure and the size of the recombinant protein indicated that Cl-IDH was a monomeric enzyme of 80 kDa distinct from the dimeric NADP-dependent IDHs predominantly found in bacteria or eukaryotic mitochondria. Apparent Michaelis constants for isocitrate (45 +/- 13 microm) and NADP+ (27 +/- 10 microm) were much smaller than those for 2-oxoglutarate (1.1 +/- 0.5 mm) and CO2 (1.3 +/- 0.3 mm). No significant differences in kinetic properties were observed between Cl-IDH and the dimeric, NADP-dependent IDH from Saccharomyces cerevisiae (Sc-IDH) at the optimum pH of each enzyme. However, in contrast to the 20% activity of Sc-IDH toward carboxylation as compared with that toward decarboxylation at pH 7.0, the activities of Cl-IDH for both directions were almost equivalent at this pH, suggesting a more favorable property of Cl-IDH than Sc-IDH as a CO2-fixation enzyme under physiological pH. Furthermore, we found that among various intermediates, oxaloacetate was a competitive inhibitor (K(i) = 0.35 +/- 0.04 mm) for 2-oxoglutarate in the carboxylation reaction by Cl-IDH, a feature not found in Sc-IDH.