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

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<jats:p>Isocitrate dehydrogenase (IDH) catalyzes the reversible conversion between isocitrate and 2‐oxoglutarate accompanied by decarboxylation/carboxylation and oxidoreduction of NAD(P)<jats:sup>+</jats:sup> 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 <jats:italic>Chlorobium limicola</jats:italic>, a green sulfur bacterium that fixes CO<jats:sub>2</jats:sub> through the reductive tricarboxylic acid (RTCA) cycle, focusing on the CO<jats:sub>2</jats:sub>‐fixation ability of the enzyme. The gene encoding <jats:italic>Cl</jats:italic>‐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 <jats:italic>Cl</jats:italic>‐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 µ<jats:sc>m</jats:sc>) and NADP<jats:sup>+</jats:sup> (27 ± 10 µ<jats:sc>m</jats:sc>) were much smaller than those for 2‐oxoglutarate (1.1 ± 0.5 m<jats:sc>m</jats:sc>) and CO<jats:sub>2</jats:sub> (1.3 ± 0.3 m<jats:sc>m</jats:sc>). No significant differences in kinetic properties were observed between <jats:italic>Cl</jats:italic>‐IDH and the dimeric, NADP‐dependent IDH from <jats:italic>Saccharomyces cerevisiae</jats:italic> (<jats:italic>Sc</jats:italic>‐IDH) at the optimum pH of each enzyme. However, in contrast to the 20% activity of <jats:italic>Sc</jats:italic>‐IDH toward carboxylation as compared with that toward decarboxylation at pH 7.0, the activities of <jats:italic>Cl</jats:italic>‐IDH for both directions were almost equivalent at this pH, suggesting a more favorable property of <jats:italic>Cl</jats:italic>‐IDH than <jats:italic>Sc</jats:italic>‐IDH as a CO<jats:sub>2</jats:sub>‐fixation enzyme under physiological pH. Furthermore, we found that among various intermediates, oxaloacetate was a competitive inhibitor (<jats:italic>K</jats:italic><jats:sub>i</jats:sub> = 0.35 ± 0.04 m<jats:sc>m</jats:sc>) for 2‐oxoglutarate in the carboxylation reaction by <jats:italic>Cl</jats:italic>‐IDH, a feature not found in <jats:italic>Sc</jats:italic>‐IDH.</jats:p>

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