Type 2 isopentenyl diphosphate isomerase from a thermoacidophilic archaeon Sulfolobus shibatae.
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.
Yamashita
Satoshi
S
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Miyagi, Japan.
Hemmi
Hisashi
H
Ikeda
Yosuke
Y
Nakayama
Toru
T
Nishino
Tokuzo
T
D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019748:Carbon-Carbon Double Bond Isomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000378:metabolism; D045782:Hemiterpenes / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009245:NADH Dehydrogenase / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D016944:Sulfolobus / Q000201:enzymology*
D001051:Apoenzymes; D019843:Archaeal Proteins; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; C043060:3,3-dimethylallyl pyrophosphate; D009249:NADP; D005486:Flavin Mononucleotide; D006861:Hydrogen Peroxide; D009245:NADH Dehydrogenase; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase; D008274:Magnesium