title: Cloning and functional characterization of β-phellandrene synthase from Lavandula angustifolia.
abstract: En route to building genomics resources for Lavandula, we have obtained over 14,000 ESTs for leaves and flowers of L. angustifolia, a major essential oil crop, and identified a number of previously uncharacterized terpene synthase (TPS) genes. Here we report the cloning, expression in E. coli, and functional characterization of β-phellandrene synthase, LaβPHLS. The ORF--excluding the transit peptide--for this gene encoded a 62.3 kDa protein that contained all conserved motifs present in plant TPSs. Expression in bacteria resulted in the production of a soluble protein that was purified by Ni-NTA agarose affinity chromatography. While the recombinant LaβPHLS did not utilize FPP as a substrate, it converted GPP (the preferred substrate) and NPP into β-phellandrene as the major product, with K (m) and k (cat) of 6.55 μM and 1.75 × 10(-2) s(-1), respectively, for GPP. The LaβPHLS transcripts were highly abundant in young leaves where β-phellandrene is produced, but were barely detectable in flowers and older leaves, where β-phellandrene is not synthesized in significant quantities. This data indicate that β-phellandrene biosynthesis is transcriptionally and developmentally regulated. We also cloned and expressed in E. coli a second TPS-like protein, LaTPS-I, that lacks an internal stretch of 73 amino acids, including the signature DDxxD divalent metal binding motif, compared to other plant TPSs. The recombinant LaTPS-I did not produce detectable products in vitro when assayed with GPP, NPP or FPP as substrates. The lack of activity is most likely due to the absence of catalytically important amino acid residues within the missing region.
authors.affiliation: Department of Biology, University of British Columbia-Okanagan, 3333 University Way, Kelowna, British Columbia, V1V 1V7, Canada.
authors.forename: Zerihun A
authors.initials: ZA
authors.lastname: Demissie
authors.forename: Lukman S
authors.initials: LS
authors.lastname: Sarker
authors.forename: Soheil S
authors.initials: SS
authors.lastname: Mahmoud
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D000081005:Cyclohexane Monoterpenes; D053138:Cyclohexenes / Q000737:chemistry / Q000378:metabolism*; D057075:Enzyme Assays; D008401:Gas Chromatography-Mass Spectrometry; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D027523:Lavandula / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D039821:Monoterpenes / Q000737:chemistry / Q000378:metabolism*; D009822:Oils, Volatile / Q000032:analysis; D010802:Phylogeny; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D014158:Transcription, Genetic
chemical_list: D000081005:Cyclohexane Monoterpenes; D053138:Cyclohexenes; D039821:Monoterpenes; D009822:Oils, Volatile; C058582:beta-phellandrene; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase