Nondecarboxylating and decarboxylating isocitrate dehydrogenases: oxalosuccinate reductase as an ancestral form of isocitrate dehydrogenase

Abstract

Isocitrate dehydrogenase (ICDH) from Hydrogenobacter thermophilus catalyzes the reduction of oxalosuccinate, which corresponds to the second step of the reductive carboxylation of 2-oxoglutarate in the reductive tricarboxylic acid cycle. In this study, the oxidation reaction catalyzed by H. thermophilus ICDH was kinetically analyzed. As a result, a rapid equilibrium random-order mechanism was suggested. The affinities of both substrates (isocitrate and NAD+) toward the enzyme were extremely low compared to other known ICDHs. The binding activities of isocitrate and NAD+ were not independent; rather, the binding of one substrate considerably promoted the binding of the other. A product inhibition assay demonstrated that NADH is a potent inhibitor, although 2-oxoglutarate did not exhibit an inhibitory effect. Further chromatographic analysis demonstrated that oxalosuccinate, rather than 2-oxoglutarate, is the reaction product. Thus, it was shown that H. thermophilus ICDH is a nondecarboxylating ICDH that catalyzes the conversion between isocitrate and oxalosuccinate by oxidation and reduction. This nondecarboxylating ICDH is distinct from well-known decarboxylating ICDHs and should be categorized as a new enzyme. Oxalosuccinate-reducing enzyme may be the ancestral form of ICDH, which evolved to the extant isocitrate oxidative decarboxylating enzyme by acquiring higher substrate affinities.

FIG. 1.

Physiological roles of ICDH in E. coli and in H. thermophilus. (A) EcICDH is an enzyme involved in the TCA cycle and catalyzes the oxidative decarboxylation of isocitrate to form 2-oxoglutarate. (B) HtICDH is an enzyme involved in the reductive TCA cycle and catalyzes oxalosuccinate reduction, which corresponds to the second step of the reductive carboxylation of 2-oxoglutarate.

FIG. 2.

Reductive carboxylation catalyzed by ICDH. Chromatograms of the mixtures obtained after the reductive carboxylation reaction by EcICDH (incubated for 5 min at 37°C using 7.3 μg of protein) (A), CnICDH (incubated for 5 min at 70°C using 6.2 μg of protein) (B), and HtICDH (incubated for 5 min at 70°C using 16 μg of protein) (C). Reactions were performed in a volume of 400 μl containing 100 mM bicine-KOH (pH 6.5), 10 mM MgCl₂, 20 mM 2-oxoglutarate, 50 mM NaHCO₃, 5 mM NADPH (for EcICDH and CnICDH) or 5 mM NADH (for HtICDH), and ICDH. After incubation, the reaction mixtures were diluted with water (threefold dilution) before injection onto the column.

FIG. 3.

Kinetics of the oxidation reaction catalyzed by HtICDH. (A) Double-reciprocal plots obtained by varying the isocitrate concentration at several different fixed concentrations of NAD⁺ (circle, 4 mM; square, 3 mM; triangle, 2 mM). (B) Double-reciprocal plots obtained by varying the NAD⁺ concentration under several different fixed concentrations of dl-isocitrate (circle, 4 mM; square, 3 mM; triangle, 2 mM). (C) Inhibition by NADH versus isocitrate. The concentration of NAD⁺ was fixed at 4 mM, and the dl-isocitrate concentration was varied at several different concentrations of NADH (circle, 0 mM; square, 0.01 mM; triangle, 0.02 mM). (D) Inhibition by NADH versus NAD⁺. The concentration of dl-isocitrate was fixed at 4 mM, and NAD⁺ concentration was varied at several different concentrations of NADH (circle, 0 mM; square, 0.01 mM; triangle, 0.02 mM). v, velocity.

FIG. 4.

Oxalosuccinate formation by the oxidation reaction. Chromatograms of the mixture obtained after the oxidation reaction by EcICDH (using 18 μg of protein) (A) and HtICDH (using 79 μg of protein) (B). The reaction was performed in a volume of 400 μl containing 100 mM bicine-KOH (pH 9.5), 10 mM MgCl₂, 2 mM dl-isocitrate, 2 mM NADP⁺ (for EcICDH) or 2 mM NAD⁺ (for HtICDH), and ICDH. After incubation at 20°C for 20 min, the reaction mixture was diluted with water (12-fold in A and 2-fold in B) before injection onto the column.

FIG. 5.

Oxidation reaction catalyzed by HtICDH. HtICDH only catalyzes the oxidation of isocitrate to oxalosuccinate. The following decarboxylation is a nonenzymatic reaction.

FIG. 6.

Proposed kinetic mechanism of the oxidation reaction catalyzed by HtICDH. Enz, HtICDH; IC, isocitrate; filled circle, isocitrate binding site of the binary complex (high affinity); open circle, isocitrate binding site of HtICDH (low affinity); filled star, NAD binding site of the binary complex (high affinity); open star, NAD binding site of HtICDH (low affinity). Arrows with dotted lines indicate product inhibition by NADH.

FIG. 7.

Hypothetical evolutionary pathway from an oxalosuccinate-reducing enzyme to an isocitrate-oxidative decarboxylating enzyme. The affinities of oxalosuccinate, isocitrate, and 2-oxoglutarate toward the enzyme are shown. H. thermophilus ICDH, an oxalosuccinate-reducing enzyme, is proposed to have evolved to an isocitrate-oxidative decarboxylating enzyme (E. coli ICDH) by acquiring higher substrate affinities.