The archaeal leuB gene encoding isopropylmalate dehydrogenase of Sulfolobus sp. strain 7 was cloned, sequenced, and expressed in Escherichia coli. The recombinant Sulfolobus sp. enzyme was extremely stable to heat. The substrate and coenzyme specificities of the archaeal enzyme resembled those of the bacterial counterparts. Sedimentation equilibrium analysis supported an earlier proposal that the archaeal enzyme is homotetrameric, although the corresponding enzymes studied so far have been reported to be dimeric. Phylogenetic analyses suggested that the archaeal enzyme is homologous to mitochondrial NAD-dependent isocitrate dehydrogenases (which are tetrameric or octameric) as well as to isopropylmalate dehydrogenases from other sources. These results suggested that the present enzyme is the most primitive among isopropylmalate dehydrogenases belonging in the decarboxylating dehydrogenase family.3-Isopropylmalate dehydrogenase (IPMDH; EC 1.1.1.85) is the third enzyme in the leucine biosynthetic pathway and catalyzes the oxidative decarboxylation of threo-D S -3-isopropylmalate to 2-oxoisocaproate (32). Genes coding for the enzyme have been cloned and sequenced from various bacteria (eubacteria) and eukarya (eukaryotes). IPMDHs so far investigated are NAD dependent and homodimeric (20,39).It has been suggested that the catalytic mechanism of IPMDH is similar to that of bacterial homodimeric NADPdependent isocitrate dehydrogenase (ICDH; EC 1.1.1.42) a key enzyme in the tricarboxylic acid cycle. The latter catalyzes the oxidative decarboxylation of threo-D S -isocitrate to oxoglutarate (9). The primary structures of IPMDHs are homologous to those of the homodimeric bacterial NADP-dependent ICDHs (42). In addition, IPMDHs show sequence similarities to mitochondrial NAD-dependent ICDHs. The eukaryotic NAD-dependent ICDHs are either heterotetrameric (e.g., ␣2␤␥ for the mammalian mitochondrial enzymes) (33, 41) or heterooctameric (e.g., ␣4␤4 for the yeast mitochondrial enzyme) (21), consisting of subunits homologous to each other.Recently, the X-ray crystal structure of Thermus thermophilus HB8 IPMDH has been determined (13,15,19). The structure resembles that of Escherichia coli 14). In particular, both enzymes are unique in lacking the Rossmann fold motif which is frequently found in NAD(P)-dependent enzymes. In addition, most of the functional key residues proposed by the crystallographic studies are conserved among IPMDHs, bacterial NADP-dependent ICDHs, and mitochondrial NAD-dependent ICDHs (42). The structural evidence strongly supports the idea (13, 42) that these enzymes originated from a gene duplication that preceded the divergence of the three domains of life.To clarify the phylogenetic relationship among IPMDHs and ICDHs, the structural and functional data of archaeal enzyme are absolutely required, since Archaea (archaebacteria) comprises the third independent domain of life, which is only distantly related to both the Bacteria and the Eukarya (38). Recently, we have isolated and preliminarily characterize...