Abstract
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°C and an apparent midpoint melting temperature (T m) of 70°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.
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Acknowledgments
This work was supported by the Norwegian Research Council (Project no. 153774/420). The Norwegian Structural Biology Centre (NORSTRUCT) is supported by the national Functional Genomics Programme (FUGE) of the Research Council of Norway. We are grateful to Dr. Aurora Martinez, Department of Biomedicine, University of Bergen, for access to her laboratory facilities and expertise in the use of differential scanning calorimetry. The excellent laboratory skills of Lisbeth Glærum and Marit Steine Madsen are also much appreciated.
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Stokke, R., Madern, D., Fedøy, AE. et al. Biochemical characterization of isocitrate dehydrogenase from Methylococcus capsulatus reveals a unique NAD+-dependent homotetrameric enzyme. Arch Microbiol 187, 361–370 (2007). https://doi.org/10.1007/s00203-006-0200-y
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DOI: https://doi.org/10.1007/s00203-006-0200-y