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Journal of Bacteriology, March 2008, p. 2050-2055, Vol. 190, No. 6
0021-9193/08/$08.00+0     doi:10.1128/JB.01799-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Nondecarboxylating and Decarboxylating Isocitrate Dehydrogenases: Oxalosuccinate Reductase as an Ancestral Form of Isocitrate Dehydrogenase{triangledown}

Miho Aoshima* and Yasuo Igarashi

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

Received 14 November 2007/ Accepted 3 January 2008

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.

* Corresponding author. Mailing address: Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8567, Japan. Phone: 81 3 5841 5143. Fax: 81 3 5841 5272. E-mail: aomiho{at}mail.ecc.u-tokyo.ac.jp

{triangledown} Published ahead of print on 18 January 2008.

Journal of Bacteriology, March 2008, p. 2050-2055, Vol. 190, No. 6
0021-9193/08/$08.00+0     doi:10.1128/JB.01799-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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