Farnesyl pyrophosphate synthase from white lupin: molecular cloning, expression, and purification of the expressed protein.
Plants produce a variety of sesquiterpenoid compounds with diverse biological functions, whose synthesis is initiated by farnesyl pyrophosphate synthase [EC 2.5.1.1, EC 2.5.1.10]. The lack of availability of molecular tools to analyze this enzyme has, thus far, prevented the study of its expression and regulation in plants. A DNA fragment corresponding to a portion of the farnesyl pyrophosphate synthase gene was amplified by the polymerase chain reaction using was amplified by the polymerase chain reaction using degenerate primers designed from two highly conserved domains (FLV(A/L)DD(I/M)MD and FQIQDDYLD) found in eukaryotic farnesyl pyrophosphate synthase sequences. A clone, pS19, of a 438-bp PCR fragment was used to screen a white lupin root cDNA library. Two full-length cDNA clones (pFPS1 and pFPS2) were isolated and sequenced, and one of them (pFPS2) was expressed in a bacterial system and the enzyme protein encoded by the clone was purified. The 1345-bp insert of pFPS2 contains a 1026-bp open reading frame, encoding a 342-amino-acid peptide with a calculated molecular mass of 39,310 Da. The deduced amino acid sequence of lupin farnesyl pyrophosphate synthase pFPS2 shares 90 and 79% identity with those from Lupinus albus (pFPS1) and Arabidopsis thaliana, respectively, 51% with the yeast enzyme, and 44% identity with those from rat and human. The overexpressed protein, which was purified to near homogeneity, displayed both dimethylallyl transferase and geranyl transferase activities. Polyclonal antibodies raised against the purified protein immunorecognized a ca 39-kDa protein in lupin root extracts.
Attucci
S
S
Department of Biology, Concordia University, Montreal, Québec, Canada.
Aitken
S M
SM
Gulick
P J
PJ
Ibrahim
R K
RK
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D017124:Conserved Sequence; D018076:DNA, Complementary; D018744:DNA, Plant / Q000378:metabolism; D007887:Fabaceae / Q000201:enzymology*; D015870:Gene Expression; D015723:Gene Library; D017343:Genes, Plant; D051228:Geranyltranstransferase; D006801:Humans; D008969:Molecular Sequence Data; D018517:Plant Roots; D010946:Plants, Medicinal; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D014166:Transferases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D018076:DNA, Complementary; D018744:DNA, Plant; D011994:Recombinant Proteins; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051228:Geranyltranstransferase