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

Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8567, Japan

^* To whom correspondence should be addressed. Email: aomiho{at}mail.ecc.u-tokyo.ac.jp.

	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 as compared to other known ICDHs. The binding 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 non-decarboxylating ICDH that catalyzes the conversion between isocitrate and oxalosuccinate by oxidation and reduction. This non-decarboxylating 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.