FARNESYL PYROPHOSPHATE SYNTHASE FROM WHITE LUPIN - MOLECULAR-CLONING,EXPRESSION, AND PURIFICATION OF THE EXPRESSED PROTEIN

Plants produce a variety of sesquiterpenoid compounds with diverse bio logical functions, whose synthesis is initiated by farnesyl pyrophosph ate synthase [EC 2.5.1.1, EC 2.5.1.10], The lack of availability of mo lecular tools to analyze this enzyme has, thus far, prevented the stud y of its expression and regulation in plants, A DNA fragment correspon ding to a portion of the farnesyl pyrophosphate synthase gene was ampl ified by the polymerase chain reaction using degenerate primers design ed from two highly conserved domains (FLV(A/L)DD(I/M)MD and FQIQDD-DYL D) found in eukaryotic farnesyl pyrophosphate synthase sequences, A cl one, pS19, of a 438-bp PCR fragment was used to screen a white lupin r oot cDNA library, Two full-length cDNA clones (pFPSI and pFPS2) were i solated and sequenced, and one of them (pFPS2) was expressed in a bact erial system and the enzyme protein encoded by the clone was purified, The 1345-bp insert of pFPS2 contains a 1026-bp open reading frame, en coding a 342-amino-acid peptide with a calculated molecular mass of 39 ,310 Da. The deduced amino acid sequence of lupin farnesyl pyrophospha te synthase pFPS2 shares 90 and 79% identity with those from Lupinus a lbus (pFPSI) and Arabidopsis thaliana, respectively, 51% with the yeas t enzyme, and 44% identity with those from rat and human, The overexpr essed protein, which was purified to near homogeneity, displayed both dimethylalkyl transferase and geranyl transferase activities, Polyclon al antibodies raised against the purified protein immunorecognized a c a 39-kDa protein in lupin root extracts. (C) 1995 Academic Press, Inc.