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

Tadayoshi Kanao, Mineko Kawamura, Toshiaki Fukui, Haruyuki Atomi, Tadayuki Imanaka

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

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 μM) and NADP+ (27 ± 10 μM) 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 (Ki = 0.35 ± 0.04 mM) for 2-oxoglutarate in the carboxylation reaction by Cl-IDH, a feature not found in Sc-IDH.

Original languageEnglish
Pages (from-to)1926-1931
Number of pages6
JournalEuropean Journal of Biochemistry
Volume269
Issue number7
DOIs
Publication statusPublished - 2002
Externally publishedYes

Fingerprint

Chlorobium
Chlorobi
Isocitrate Dehydrogenase
Citric Acid Cycle
Sulfur
Carbon Dioxide
Bacteria
Enzymes
Carboxylation
NADP
Decarboxylation
Oxaloacetic Acid
Recombinant Proteins
NAD
Mitochondria
Gene encoding
Saccharomyces cerevisiae

Keywords

  • CO-fixing enzyme
  • Isocitrate dehydrogenase
  • Reductive tricarboxylic acid cycle

ASJC Scopus subject areas

  • Biochemistry

Cite this

Characterization of isocitrate dehydrogenase from the green sulfur bacterium chlorobium limicola : A carbon dioxide-fixing enzyme in the reductive tricarboxylic acid cycle. / Kanao, Tadayoshi; Kawamura, Mineko; Fukui, Toshiaki; Atomi, Haruyuki; Imanaka, Tadayuki.

In: European Journal of Biochemistry, Vol. 269, No. 7, 2002, p. 1926-1931.

Research output: Contribution to journalArticle

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abstract = "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 μM) and NADP+ (27 ± 10 μM) 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 (Ki = 0.35 ± 0.04 mM) for 2-oxoglutarate in the carboxylation reaction by Cl-IDH, a feature not found in Sc-IDH.",
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T2 - A carbon dioxide-fixing enzyme in the reductive tricarboxylic acid cycle

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AU - Kawamura, Mineko

AU - Fukui, Toshiaki

AU - Atomi, Haruyuki

AU - Imanaka, Tadayuki

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AB - 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 μM) and NADP+ (27 ± 10 μM) 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 (Ki = 0.35 ± 0.04 mM) for 2-oxoglutarate in the carboxylation reaction by Cl-IDH, a feature not found in Sc-IDH.

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