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Although isopentenyl diphosphate–dimethylallyl diphos-phate isomerase is thought to be essential for archaea because they use themevalonate pathway, its corresponding activityhas not beendetected inanyarchaea.Anovel typeof the enzyme, which has no sequence similarity to the known, well-studied type of enzymes, was recently reported in some bacterial strains. In this study, we describe the cloning of a gene of a homologue of the novel bacterial isomerase froma thermoacidophilic archaeon Sulfolobus shibatae. . | Eur. J. Biochem. 271 1087-1093 2004 FEBS 2004 doi 10.1111 j.1432-1033.2004.04010.x Type 2 isopentenyl diphosphate isomerase from a thermoacidophilic archaeon Sulfolobus shibatae Satoshi Yamashita Hisashi Hemmi Yosuke Ikeda Toru Nakayama and Tokuzo Nishino Department of Biomolecular Engineering Graduate School of Engineering Tohoku University Miyagi Japan Although isopentenyl diphosphate-dimethylallyl diphosphate isomerase is thought to be essential for archaea because they use the mevalonate pathway its corresponding activity has not been detected in any archaea. A novel type of the enzyme which has no sequence similarity to the known well-studied type of enzymes was recently reported in some bacterial strains. In this study we describe the cloning of a gene of a homologue of the novel bacterial isomerase from a thermoacidophilic archaeon Sulfolobus shibatae. The gene was heterologously expressed in Escherichia coli and the recombinant enzyme was purified and characterized. The thermostable archaeal enzyme is tetrameric and requires NAD P H and Mg2 for activity similar to its bacterial homologues. Using its apoenzyme we were able to confirm that the archaeal enzyme is strictly dependent on FMN. Moreover we provide evidence to show that the enzyme also has NADH dehydrogenase activity although it catalyzes the isomerase reaction without consuming any detectable amount of NADH. Keywords isopentenyl diphosphate-dimethylallyl diphosphate isomerase isoprenoid archaea flavoprotein NADH dehydrogenase. Isoprenoid compounds are the most diverse family of metabolites found in nature. They are necessary for all living organisms because they are functional parts of important compounds including vitamins hormones respiratory quinones and archaeal membranes 1 . The majority of isoprenoid compounds are synthesized from linear prenyl diphosphates which are formed via the consecutive condensation of isopentenyl diphosphate IPP the active isoprene C5-unit to its highly electrophilic