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.

pH‐controlled reaction divergence of decarboxylation versus fragmentation in reactions of dihydroxyfumarate with glyoxylate and formaldehyde: parallels to biological pathways

“… Comparison with enzyme‐mediated reactions (Scheme ): The fragmentation or “splitting” reaction (deoxalation) of intermediate 3 in Scheme and of intermediate 14 in Scheme at high pH has interesting parallels with similar reactions in metabolic pathways (Schemes and ). For example, oxalosuccinate (Scheme ) is an important intermediate in the citric acid cycle and is formed from isocitrate mediated by the enzyme isocitrate dehydrogenase . The structure of the tricarboxylic acid intermediate 3 (Scheme ) has an interesting resemblance to oxalosuccinate (a central intermediate in the citric and reverse‐citric acid cycle ) and its enzymatic reactions: (a) decarboxylation of oxalosuccinate to yield glutarate and (b) a proposed “splitting of oxalate” of oxalosuccinate to yield oxalate and succinate . Intermediate 3 is nothing but the 2,3‐bis‐hydroxylated analog of oxalosuccinate (Scheme ).…”

Ecological Aspects of the Distribution of Different Autotrophic CO 2 Fixation Pathways

“…Consequently, heat-labile succinyl-CoA (24) forms a large pool and cannot be effectively removed and trapped in stabile intermediates of the pathway. Aquificae (as studied in Hydrogenobacter thermophilus) solve this problem by investing additional ATP in the conversion of 2-oxoglutarate to isocitrate by the combined action of an irreversible biotin-dependent 2-oxoglutarate carboxylase and a nondecarboxylating isocitrate dehydrogenase (3,6). Together with low K m values of 2-oxoglutarate synthase for succinyl-CoA (125), this probably makes the process reasonably effective and irreversible at elevated temperatures.…”

“…Since reduced ferredoxin bears more energy than NADPH (ferredoxin, E 0 Ј Ϸ Ϫ400 mV; NADPH, E 0 Ј ϭ Ϫ320 mV), aerobic pathways usually require more ATP equivalents than anaerobic ones. A carboxylating enzyme links either CO 2 or HCO 3 Ϫ with an organic acceptor molecule, which must be regenerated in the following steps of the pathway. These inorganic carbon species are related by the pH-dependent equilibrium CO 2 ϩ H 2 O 7 H 2 CO 3 7 H ϩ ϩ HCO 3 Ϫ 7 2H ϩ ϩ CO 3…”

Isocitrate Dehydrogenase from Streptococcus mutans: Biochemical Properties and Evaluation of a Putative Phosphorylation Site at Ser102

“…SmIDH was found to have an optimal pH range of 7.5–8.5, with the optimal pH at 7.8 (Figure 2A), lower than that of the NAD + -IDHs from A . thiooxidans (pH 8.5) [2] and Hydrogenobacter thermophilus (pH 10.5) [38]. When compared with the broad optimum pH range of NADP + -IDHs from other sources, such as Streptomyces lividans (pH 8.5–10.0) [39], Fomitopsis palustri s (pH 8.0–10.0) [40] and Aspergillus niger (pH 6.0–8.0) [41], it was narrower for SmIDH, suggesting that SmIDH was sensitive to pH changes.…”