Abstract
Farnesyl diphosphate synthase catalyzes the sequential head-to-tail condensation of two molecules of isopentenyl diphosphate with dimethylallyl diphosphate. In plants the presence of farnesyl diphosphate synthase isozymes offers the possibility of differential regulation. Three full-length cDNAs encoding putative isoprenoid synthases, FDS-1, FDS-2, and FDS-5, with greater than 89% similarity were isolated from a Big Sagebrush Artemisia tridentata cDNA library using a three-step polymerase chain reaction protocol. One of the open reading frames, FDS-5, encoded a protein with an N-terminal amino acid extension that was identified as a plastidial targeting peptide. Recombinant histidine-tagged versions of three proteins were purified, and their enzymatic properties were characterized. FDS-1 and FDS-2 synthesized farnesyl diphosphate as the final chain elongation product, but their kinetic behavior varied. FDS-1 prefers geranyl diphosphate over dimethylallyl diphosphate as an allylic substrate and is active at acidic pH values compared with FDS-2. In contrast, FDS-5 synthesized two irregular monoterpenoids, chrysanthemyl diphosphate and lavandulyl diphosphate, when incubated with dimethylallyl diphosphate and an additional product, the regular monoterpene geranyl diphosphate, when incubated with isopentenyl diphosphate and dimethylallyl diphosphate. Specific cellular functions are proposed for each of the three enzymes, and a scenario for evolution of isoprenyl synthases in plants is presented.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY308477 (FDS-1), AY308476 (FDS-2), and AY308478 (ArtCDS)
The mevalonate pathway for the synthesis of isopentenyl diphosphate (IPP) and DMAPP is located in the cytosol where the triterpenoids, including phytosterols, sesquiterpenoids, and prenylated proteins, are synthesized, whereas the 2-C-methyl-D-erythritol 4-phosphate pathway operates in plant plastids, where isoprenoids involved in photosynthesis and many secondary metabolites are synthesized [4]
We describe the isolation of genes for two isoforms of farnesyl diphosphate synthase (FDS) and a gene for chrysanthemyl diphosphate synthase (CDS) from an A. tridentata ssp. spiciformis cDNA library
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY308477 (FDS-1), AY308476 (FDS-2), and AY308478 (ArtCDS). The mevalonate pathway for the synthesis of IPP and DMAPP is located in the cytosol where the triterpenoids, including phytosterols, sesquiterpenoids, and prenylated proteins, are synthesized, whereas the 2-C-methyl-D-erythritol 4-phosphate pathway operates in plant plastids, where isoprenoids involved in photosynthesis (carotenoids, chlorophyll, and plastoquinone) and many secondary metabolites are synthesized [4] Mitochondrial isoprenoids, such as ubiquinones, are synthesized directly in the mitochondria from IPPs imported from the cytosol [5]. The presence of isogenes coupled with the fact that FPP is located at a multiple branch point in the isoprenoid biosynthetic pathway suggests that FPP biosynthesis is tightly regulated Evidence for this hypothesis was obtained by Cunillera et al, who demonstrated that Arabidopsis thaliana contains at least two differentially regulated genes coding for FDS [9] and later showed that one of these genes is controlled at the transcriptional level. Use cDNA preparation cDNA preparation First-step PCR First-step PCR First-step PCR First-step PCR Third-step PCR Second-step PCR P3 for FDS-1 cloning P4 for FDS-1 cloning P3 for FDS-2 cloning P4 for FDS-2 cloning P3 for FDS-5 cloning P4 for FDS-5 cloning GFP fusion protein GFP fusion protein Enzyme expression Enzyme expression Enzyme expression
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