cDNA cloning, characterization, and functional expression of four new monoterpene synthase members of the Tpsd gene family from grand fir (Abies grandis)

Grand fir (Abies grandis) is a useful model system for studying the biochem istry, molecular genetics, and regulation of defensive oleoresin formation in conifers, a process involving both the constitutive accumulation of resi n (pitch) in specialized secretory structures and the induced biosynthesis of monoterpenes and sesquiterpenes (turpentine) and diterpene resin acids ( rosin) by nonspecialized cells at the site of injury. A similarity-based cl oning strategy, employing primers designed to conserved regions of existing monoterpene synthases and anticipated to amplify a 1000-bp fragment, unexp ectedly yielded a 300-bp fragment with sequence reminiscent of a terpenoid synthase. Utilization of this amplicon as a hybridization probe afforded fo ur new, full-length cDNA species from a wounded fir stem cDNA library that appeared to encode four distinct monoterpene synthases. Expression in Esche richia coli, followed by enzyme assay with geranyl diphosphate (C-10), farn esyl diphosphate (C-15) and geranylgeranyl diphosphate (C-20), and analysis of the terpene products by chiral phase gas chromatography and mass spectr ometry confirmed that these sequences encoded four new monoterpene synthase s, including (-)-camphene synthase, (-)-beta-phellandrene synthase, terpino lene synthase, and an enzyme that produces both (-)-limonene and (-)-alpha- pinene. The deduced amino acid sequences indicated these enzymes to be 618 to 637 residues in length (71 to 73 kDa) and to be translated as preprotein s bearing an amino-terminal plastid targeting sequence of 50-60 residues. c DNA truncation to delete the transit peptide allowed functional expression of the "pseudomature" forms of these enzymes, which exhibited no change in product outcome as a result of truncation. Sequence comparison revealed tha t these new monoterpene synthases from grand fir are members of the Tpsd ge ne subfamily and resemble sesquiterpene (C-15) synthases and diterpene (C-2 0) synthases from conifers more closely than mechanistically related monote rpene synthases from angiosperm species. The availability of a nearly compl ete set of constitutive and inducible monoterpene synthases from grand fir (now numbering seven) will allow molecular dissection of the resin-based de fense response in this conifer species, and detailed study of structure-fun ction relationships among this large and diverse family of catalysts, all o f which exploit the same stereochemistry in the coupled isomerization-cycli zation reaction. (C) 1999 Academic Press.