title: Localization and in-Vivo Characterization ofThapsia garganicaCYP76AE2 Indicates a Role in Thapsigargin Biosynthesis.
abstract: The Mediterranean plantThapsia garganica(dicot, Apiaceae), also known as deadly carrot, produces the highly toxic compound thapsigargin. This compound is a potent inhibitor of the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase calcium pump in mammals and is of industrial importance as the active moiety of the anticancer drug mipsagargin, currently in clinical trials. Knowledge of thapsigargin in planta storage and biosynthesis has been limited. Here, we present the putative second step in thapsigargin biosynthesis, by showing that the cytochrome P450 TgCYP76AE2, transiently expressed inNicotiana benthamiana, converts epikunzeaol into epidihydrocostunolide. Furthermore, we show that thapsigargin is likely to be stored in secretory ducts in the roots. Transcripts from TgTPS2 (epikunzeaol synthase) and TgCYP76AE2 in roots were found only in the epithelial cells lining these secretory ducts. This emphasizes the involvement of these cells in the biosynthesis of thapsigargin. This study paves the way for further studies of thapsigargin biosynthesis.
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, 1871 Frederiksberg C, Denmark (T.B.A., K.J., J.A.-R.).|Natural History Museum of Denmark, University of Copenhagen, DK-1350 Copenhagen K, Denmark (K.A.M.).|Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, 2800 Kgs. Lyngby, Denmark (K.A.M., S.A.R., H.T.S.).|Metabolomics Australia, School of BioSciences, University of Melbourne, Melbourne, Victoria 3010, Australia (B.A.B.); and.|Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark (N.N., S.B.C.).
authors.forename: Trine Bundgaard
authors.initials: TB
authors.lastname: Andersen
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, 1871 Frederiksberg C, Denmark (T.B.A., K.J., J.A.-R.).|Natural History Museum of Denmark, University of Copenhagen, DK-1350 Copenhagen K, Denmark (K.A.M.).|Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, 2800 Kgs. Lyngby, Denmark (K.A.M., S.A.R., H.T.S.).|Metabolomics Australia, School of BioSciences, University of Melbourne, Melbourne, Victoria 3010, Australia (B.A.B.); and.|Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark (N.N., S.B.C.).
authors.forename: Karen Agatha
authors.initials: KA
authors.lastname: Martinez-Swatson
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, 1871 Frederiksberg C, Denmark (T.B.A., K.J., J.A.-R.).|Natural History Museum of Denmark, University of Copenhagen, DK-1350 Copenhagen K, Denmark (K.A.M.).|Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, 2800 Kgs. Lyngby, Denmark (K.A.M., S.A.R., H.T.S.).|Metabolomics Australia, School of BioSciences, University of Melbourne, Melbourne, Victoria 3010, Australia (B.A.B.); and.|Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark (N.N., S.B.C.).
authors.forename: Silas Anselm
authors.initials: SA
authors.lastname: Rasmussen
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, 1871 Frederiksberg C, Denmark (T.B.A., K.J., J.A.-R.).|Natural History Museum of Denmark, University of Copenhagen, DK-1350 Copenhagen K, Denmark (K.A.M.).|Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, 2800 Kgs. Lyngby, Denmark (K.A.M., S.A.R., H.T.S.).|Metabolomics Australia, School of BioSciences, University of Melbourne, Melbourne, Victoria 3010, Australia (B.A.B.); and.|Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark (N.N., S.B.C.).
authors.forename: Berin Alain
authors.initials: BA
authors.lastname: Boughton
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, 1871 Frederiksberg C, Denmark (T.B.A., K.J., J.A.-R.).|Natural History Museum of Denmark, University of Copenhagen, DK-1350 Copenhagen K, Denmark (K.A.M.).|Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, 2800 Kgs. Lyngby, Denmark (K.A.M., S.A.R., H.T.S.).|Metabolomics Australia, School of BioSciences, University of Melbourne, Melbourne, Victoria 3010, Australia (B.A.B.); and.|Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark (N.N., S.B.C.).
authors.forename: Kirsten
authors.initials: K
authors.lastname: Jørgensen
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, 1871 Frederiksberg C, Denmark (T.B.A., K.J., J.A.-R.).|Natural History Museum of Denmark, University of Copenhagen, DK-1350 Copenhagen K, Denmark (K.A.M.).|Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, 2800 Kgs. Lyngby, Denmark (K.A.M., S.A.R., H.T.S.).|Metabolomics Australia, School of BioSciences, University of Melbourne, Melbourne, Victoria 3010, Australia (B.A.B.); and.|Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark (N.N., S.B.C.).
authors.forename: Johan
authors.initials: J
authors.lastname: Andersen-Ranberg
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, 1871 Frederiksberg C, Denmark (T.B.A., K.J., J.A.-R.).|Natural History Museum of Denmark, University of Copenhagen, DK-1350 Copenhagen K, Denmark (K.A.M.).|Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, 2800 Kgs. Lyngby, Denmark (K.A.M., S.A.R., H.T.S.).|Metabolomics Australia, School of BioSciences, University of Melbourne, Melbourne, Victoria 3010, Australia (B.A.B.); and.|Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark (N.N., S.B.C.).
authors.forename: Nils
authors.initials: N
authors.lastname: Nyberg
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, 1871 Frederiksberg C, Denmark (T.B.A., K.J., J.A.-R.).|Natural History Museum of Denmark, University of Copenhagen, DK-1350 Copenhagen K, Denmark (K.A.M.).|Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, 2800 Kgs. Lyngby, Denmark (K.A.M., S.A.R., H.T.S.).|Metabolomics Australia, School of BioSciences, University of Melbourne, Melbourne, Victoria 3010, Australia (B.A.B.); and.|Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark (N.N., S.B.C.).
authors.forename: Søren Brøgger
authors.initials: SB
authors.lastname: Christensen
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, 1871 Frederiksberg C, Denmark (T.B.A., K.J., J.A.-R.); hets@dtu.dk.|Natural History Museum of Denmark, University of Copenhagen, DK-1350 Copenhagen K, Denmark (K.A.M.); hets@dtu.dk.|Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, 2800 Kgs. Lyngby, Denmark (K.A.M., S.A.R., H.T.S.); hets@dtu.dk.|Metabolomics Australia, School of BioSciences, University of Melbourne, Melbourne, Victoria 3010, Australia (B.A.B.); and hets@dtu.dk.|Department of Drug Design and Pharmacology, University of Copenhagen, 2100 Copenhagen, Denmark (N.N., S.B.C.) hets@dtu.dk.
authors.forename: Henrik Toft
authors.initials: HT
authors.lastname: Simonsen
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D020869:Gene Expression Profiling / Q000379:methods; D018506:Gene Expression Regulation, Plant; D008956:Models, Chemical; D015394:Molecular Structure; D010802:Phylogeny; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D018517:Plant Roots / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053629:Thapsia / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D019284:Thapsigargin / Q000138:chemical synthesis / Q000378:metabolism*; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D019284:Thapsigargin; D003577:Cytochrome P-450 Enzyme System