The gene encoding tartrate dehydrogenase has been cloned from Pseudomonas putida and sequenced. The gene is 1098 nucleotides long and encodes a protein 365 amino acids in length with a calculated M(r) of 40,636. The gene and the protein encoded by it show strong homology to prokaryotic isopropylmalate dehydrogenases and, to a lesser extent, isocitrate dehydrogenase. Thus, tartrate dehydrogenase is the third member to be identified of the family of metal-dependent decarboxylating R-hydroxyacid dehydrogenases which have an evolutionarily distinct NAD(+)-binding domain. The poor catalytic properties of tartrate dehydrogenase suggest that it has not been under strong selective pressure to maximize its ability to turn over (+)-tartrate for very long; the homology with isopropylmalate dehydrogenase makes it an attractive candidate for a recent progenitor of tartrate dehydrogenase. beta-Isopropylmalate is a substrate for tartrate dehydrogenase with a Km of 14 +/- 2 microM; it is turned over with a Vmax that is 35% of Vmax in the (+)-tartrate reaction. The gene encoding tartrate dehydrogenase has been expressed in Escherichia coli and large quantities of soluble enzyme can be obtained.