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 (Tm) 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.
Stokke
Runar
R
Department of Biology, University of Bergen, P.O. Box 7800, 5020 Bergen, Norway.
Madern
Dominique
D
Fedøy
Anita-Elin
AE
Karlsen
Solveig
S
Birkeland
Nils-Kåre
NK
Steen
Ida Helene
IH
D000429:Alcohol Oxidoreductases / Q000378:metabolism; D007521:Isocitrate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020619:Methylococcus capsulatus / Q000201:enzymology* / Q000235:genetics; D009243:NAD / Q000737:chemistry*; D010802:Phylogeny; D013379:Substrate Specificity
D009243:NAD; D000429:Alcohol Oxidoreductases; D007521:Isocitrate Dehydrogenase; C021368:homoisocitrate dehydrogenase