Cloning and kinetic characterization of Arabidopsis thaliana solanesyl diphosphate synthase

Abstract

trans-Long-chain prenyl diphosphate synthases catalyse the sequential condensation of isopentenyl diphosphate (C5) units with allylic diphosphate to produce the C30—C50 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 His6-tagged protein was purified and characterized. Product analysis revealed that the cDNA encodes solanesyl diphosphate (C45) synthase (At-SPS). At-SPS utilized farnesyl diphosphate (FPP; C15) and geranylgeranyl diphosphate (GGPP; C20), but did not accept either the C5 or the C10 allylic diphosphate as a primer substrate. The Michaelis constants for FPP and GGPP were 5.73μM and 1.61μM 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 C45 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.