title: 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.
authors.affiliation: Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8567, Japan. aomiho@mail.ecc.u-tokyo.ac.jp
authors.forename: Miho
authors.initials: M
authors.lastname: Aoshima
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Igarashi
mesh_terms: D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D007521:Isocitrate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D007523:Isocitrates / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D007523:Isocitrates; D007656:Ketoglutaric Acids; D011994:Recombinant Proteins; D009243:NAD; C034219:isocitric acid; D007521:Isocitrate Dehydrogenase