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Formate dehydrogenase (E.C. 1.2.1.2) is a mitochondrial-localized NAD-requiring enzyme in green plants. The enzyme activity and corresponding mRNA in leaves of Arabidopsis thaliana are induced by treatment with one-carbon metabolites. The cDNA for the Arabidopsis formate dehydrogenase is similar to that of other plants except for the N-terminal region, which is predicted to target chloroplasts as well as mitochondria. The specific of activity of the enzyme in isolated chloroplasts suggests it is targeted to both mitochondria and chloroplasts in Arabidopsis. Formate dehydrogenase from Arabidopsis was partially purified and K_m values for formate and NAD⁺ were determined to be 10 mM and 65 μM, respectively; the K_i for NADH was 17 μM. We conclude that formate dehydrogenase is normally present in Arabidopsis chloroplasts and that sensitivity to inhibition by NADH may play a role in whether cellular …