Cloning and kinetic characterization of Arabidopsis thaliana solanesyl diphosphate synthase.
trans -Long-chain prenyl diphosphate synthases catalyse the sequential condensation of isopentenyl diphosphate (C(5)) units with allylic diphosphate to produce the C(30)-C(50) prenyl diphosphates, which are precursors of the side chains of prenylquinones. Based on the relationship between product specificity and the region around the first aspartate-rich motif in trans -prenyl diphosphate synthases characterized so far, we have isolated the cDNA for a member of trans -long-chain prenyl diphosphate synthases from Arabidopsis thaliana. The cDNA was heterologously expressed in Escherichia coli, and the recombinant His(6)-tagged protein was purified and characterized. Product analysis revealed that the cDNA encodes solanesyl diphosphate (C(45)) synthase (At-SPS). At-SPS utilized farnesyl diphosphate (FPP; C(15)) and geranylgeranyl diphosphate (GGPP; C(20)), but did not accept either the C(5) or the C(10) allylic diphosphate as a primer substrate. The Michaelis constants for FPP and GGPP were 5.73 microM and 1.61 microM respectively. We also performed an analysis of the side chains of prenylquinones extracted from the A. thaliana plant, and showed that its major prenylquinones, i.e. plastoquinone and ubiquinone, contain the C(45) prenyl moiety. This suggests that At-SPS might be devoted to the biosynthesis of either or both of the prenylquinone side chains. This is the first established trans -long-chain prenyl diphosphate synthase from a multicellular organism.
Hirooka
Kazutake
K
Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita Yamadaoka 2-1, Osaka 565-0871, Japan.
Bamba
Takeshi
T
Fukusaki
Ei-ichiro
E
Kobayashi
Akio
A
D019883:Alkyl and Aryl Transferases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D030562:Databases, Protein; D007700:Kinetics; D008969:Molecular Sequence Data
D019883:Alkyl and Aryl Transferases; C022644:trans-octaprenyltranstransferase