Isopentenyl diphosphate (IPP) isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway. A database search based on probes from the highly conserved regions in three eukaryotic IPP isomerases revealed substantial similarity with ORF176 in the photosynthesis gene cluster in Rhodobacter capsulatus. The open reading frame was cloned into an Escherichia coli expression vector. The encoded 20-kDa protein, which was purified in two steps by ion exchange and hydrophobic interaction chromatography, catalyzed the interconversion of IPP and dimethylallyl diphosphate. Thus, the photosynthesis gene cluster encodes all of the enzymes required to incorporate IPP into the ultimate carotenoid and bacteriochlorophyll metabolites in R. capsulatus. More recent searches uncovered additional putative open reading frames for IPP isomerase in seed-bearing plants (Oryza sativa, Arabadopsis thaliana, and Clarkia breweri), a worm (Caenorhabiditis elegans), and another eubacterium (Escherichia coli). The R. capsulatus enzyme is the smallest of the IPP isomerases to be identified thus far and may consist mostly of a fundamental catalytic core for the enzyme.Isoprenoid metabolism, with more than 23,000 known products, is the most chemically diverse biosynthetic pathway in nature. Isoprenoids are found in all organisms (25) and comprise several classes of essential compounds, including sterols (18), carotenoids (12), dolichols (16), ubiquinones (4), and prenylated proteins (8). All of these metabolites are derived from linear isoprenoid diphosphates synthesized from the isomeric five-carbon precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (20). In eukarya and archaea, in which the pathway is well established, IPP is synthesized from three molecules of acetyl coenzyme A by the classical mevalonate pathway (18). However, in some bacteria, IPP is synthesized by a nonclassical route that is not yet fully characterized (21). Beyond IPP, the pathway for constructing polyisoprenoid chains is similar for organisms in all three major kingdoms.Isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IPP isomerase; EC 5.3.3.2) catalyzes a crucial activation step in the isoprenoid pathway by converting IPP to its highly electrophilic isomer, DMAPP. These two metabolites are the initial substrates for prenyltransferases that synthesize polyisoprenoid chains, as illustrated in Fig. 1. A gene encoding IPP isomerase, IDI1, was first isolated from Saccharomyces cerevisiae (2). Subsequently, we identified IDI1 in Schizosaccharomyces pombe by plasmid shuffle-mediated complementation of the disrupted yeast gene (13). Xuan et al. (29) found a human cDNA sequence in a phorbol-induced library with an open reading frame (ORF) encoding a protein with a high degree of similarity to both fungal IPP isomerases (13). All three proteins contained conserved regions surrounding two residues, corresponding to cysteine 139 and glutamate 207 in the S. cerevisiae enzyme, that were shown to be essential for catalys...