Geranylgeranyl diphosphate syntheses from Scoparia dulcis and Croton sublyratus. cDNA cloning, functional expression, and conversion to a farnesyl diphosphate synthase

cDNAs encoding geranylgeranyl diphosphate synthase (GGPPS) of two diterpene producing plants, Scoparia dulcis and Croton sublyratus, were isolated usi ng the homology-based polymerase chain reaction method. Both cloned genes s howed high amino acid sequence homology (60-70%) to other plant GGPPSs and contained highly conserved aspartate-rich motifs. The obtained clones were functionally expressed in Escherichia coli and showed sufficient GGPPS acti vity to catalyze the condensation of farnesyl diphosphate (FPP) and isopent enyl diphosphate to form geranylgeranyl diphosphate. To investigate the fac tor determining the product chain length of plant GGPPSs, S. dulcis GGPPS m utants in which either the small amino acids at the fourth and fifth positi ons before the first aspartate-rich motif (FARM) were replaced with aromati c amino acids or in which two additional amino acids in FARM were deleted w ere constructed. Both mutants behaved like FPPS-like enzymes and almost exc lusively produced FPP when dimethylallyl diphosphate was used as a primer s ubstrate, and failed to accept FPP as a primer substrate. These results ind icate that both small amino acids at the fourth and fifth positions before FARM and the amino acid insertion in FARM play essential roles in product l ength determination in plant GGPPSs.