Purification and characterization of a monomeric isocitrate dehydrogenase with dual coenzyme specificity from the photosynthetic bacterium Rhodomicrobium vannielii

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Abstract
An isocitrate dehydrogenase able to function with either NADP or NAD as coenzyme was purified to homogeneity from cell-free extracts of the purple photosynthetic eubacterium Rhodomicrobium vannielii using a rapid two-step procedure involving dye-ligand affinity chromatography. The enzyme was obtained in 60% yield with specific activities of 23 U.mg protein-1 (NADP-linked reaction) and 18.5 U.mg protein-1 (NAD-linked reaction). The purified enzyme was monomeric and migrated with an approximate Mr of 75,000-80,000 on both SDS/PAGE and non-denaturing PAGE. Affinity constants (Km values) of 2.5 microM for NADP and 0.77 mM for NAD and values for kcat/Km of 981,200 min-1.mM-1 (NADP) and 2455 min-1.mM-1 (NAD) indicated a greater specificity for NADP compared to NAD. A number of metabolites were examined for possible differential regulatory effects on the NADP- and NAD-linked reactions, using a dual-wavelength assay. Oxaloacetate was found to be an effective inhibitor of both reactions and the enzyme was also sensitive to concerted inhibition by glyoxylate and oxaloacetate. The amino-acid composition and the identity of 39 residues at the N-terminus were determined and compared to other isocitrate dehydrogenases. The results suggested a relationship between the Rm. vannielii enzyme and the monomeric isocitrate dehydrogenase isoenzyme II from Vibrio ABE-1.