Biochemical characterization of isocitrate dehydrogenase from Methylococcus capsulatus reveals a unique NAD+-dependent homotetrameric enzyme

The gene encoding isocitrate dehydrogenase (IDH) of Methylococcus capsulatus (McIDH) was cloned and overexpressed in Escherichia coli. The purified enzyme was NAD(+)-dependent with a thermal optimum for activity at 55-60 degrees C and an apparent midpoint melting temperature (T (m)) of 70 degrees C. Analytical ultracentrifugation (AUC) revealed a homotetrameric state, and McIDH thus represents the first homotetrameric NAD(+)-dependent IDH that has been characterized. Based on a structural alignment of McIDH and homotetrameric homoisocitrate dehydrogenase (HDH) from Thermus thermophilus (TtHDH), we identified the clasp-like domain of McIDH as a likely site for tetramerization. McIDH showed moreover, higher sequence identity (48%) to TtHDH than to previously characterized IDHs. Putative NAD(+)-IDHs with high sequence identity (48-57%) to McIDH were however identified in a variety of bacteria showing that NAD(+)-dependent IDHs are indeed widespread within the domain, Bacteria. Phylogenetic analysis including these new sequences revealed a close relationship with eukaryal allosterically regulated NAD(+)-IDH and the subfamily III of IDH was redefined to include bacterial NAD(+)- and NADP(+)-dependent IDHs. This apparent relationship suggests that the mitochondrial genes encoding NAD(+)-IDH are derived from the McIDH-like IDHs.