| Geranyl diphosphate synthase, which catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to geranyl diphosphate, the key precursor of monoterpene biosynthesis, was purified from isolated oil glands of spearmint. Peptide fragments, generated from pure proteins of 27 and 35 kDa, revealed amino acid sequences that matched two cDNA clones, obtained by random screening of a peppermint oil gland cDNA library, with deduced amino acid similarity to existing prenyltransferases and plastid targeting sequences. Expression of each cDNA individually yielded no detectable prenyltransferase activity; however, co-expression produced geranyl diphosphate. Antibodies raised against each protein were used to demonstrate that both subunits were required to produce catalytically-active native and recombinant geranyl diphosphate synthase.;Geranyl diphosphate synthase large subunit has high amino acid sequence identity (57--75%) to functional plant geranylgeranyl diphosphate synthases; however, the small subunit has low sequence identity (24--27%) to prenyltransferases and lacks the aspartate rich motifs required for prenyltransferase activity. Plasmids bearing the amino sequence of geranyl diphosphate synthase small subunit, with an 8-histidine chain attached to the carboxyl terminus, were co-expressed with spearmint farnesyl diphosphate synthase and Taxus geranylgeranyl diphosphate synthase in Escherichia coli . Lysate passed over a Ni-affinity matrix revealed that the geranyl diphosphate synthase small subunit bound to geranylgeranyl diphosphate synthase, creating a hybrid-dimer, and not to farnesyl diphosphate synthase. The reaction products from the hybrid-dimer were primarily geranyl diphosphate (C 10) with trace amounts of farnesyl diphosphate (C15) and geranylgeranyl diphosphate (C20), yet unlike geranyl diphosphate synthase, the hybrid-dimer accepted farnesyl diphosphate and geranylgeranyl diphosphate as allylic substrate. Results from gel permeation chromatography show that both recombinant and native geranyl diphosphate synthase from Mentha have a subunit composition that is tetrameric (4); whereas, the hybrid-dimer and geranylgeranyl diphosphate synthase had a subunit composition of two. Michaelis Menton constants (Km) and catalytic turnover rates (Kcat) reveal that the hybrid-dimer shares catalytic similarities to both geranyl diphosphate synthase and geranylgeranyl diphosphate synthase. |
