Biochemical and molecular characterization of the isocitrate dehydrogenase with dual coenzyme specificity from the obligate methylotroph Methylobacillus Flagellatus

The isocitrate dehydrogenase ( Mf IDH) with unique double coenzyme specificity from Methylobacillus flagellatus was purified and characterized, and its gene was cloned and overexpressed in E . coli as a fused protein. This enzyme is homodimeric,—with a subunit molecular mass of 45 kDa and a specific activity of 182 U mg -1 with NAD + and 63 U mg -1 with NADP + . The Mf IDH activity was dependent on divalent cations and Mn 2+ enhanced the activity the most effectively. Mf IDH exhibited a cofactor-dependent pH-activity profile. The optimum pH values were 8.5 (NAD +) and 6.0 (NADP + ).The K m values for NAD + and NADP + were 113 μM and 184 μM respectively, while the K m values for DL-isocitrate were 9.0 μM (NAD + ), 8.0 μM (NADP + ). The Mf IDH specificity (k cat /K m ) was only 5-times higher for NAD + than for NADP + . The purified Mf IDH displayed maximal activity at 60°C. Heat-inactivation studies showed that the Mf IDH was remarkably thermostable, retaining full activity at 50°C and losting ca. 50% of its activity after one hour of incubation at 75°C. The enzyme was insensitive to the presence of intermediate metabolites, with the exception of 2 mM ATP, which caused 50% inhibition of NADP + -linked activity. The indispensability of the N 6 amino group of NAD(P) + in its binding to Mf IDH was demonstrated. Mf IDH showed high sequence similarity with bacterial NAD(P) + -dependent type I isocitrate dehydrogenases (IDHs) rather than with eukaryotic NAD + -dependent IDHs. The unique double coenzyme specificity of Mf IDH potentially resulted from the Lys340, Ile341 and Ala347 residues in the coenzyme-binding site of the enzyme. The discovery of a type I IDH with double coenzyme specificity elucidates the evolution of this subfamily IDHs and may provide fundamental information for engineering enzymes with desired properties.