title: Identification and characterization of a kunzeaol synthase from Thapsia garganica: implications for the biosynthesis of the pharmaceutical thapsigargin.
abstract: Thapsigargin is a major terpenoid constituent of Thapsia garganica root. Owing to its potent antagonistic effect on the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, thapsigargin has been widely used to study Ca2+ signalling and is also a potential drug for prostate cancer. Despite its importance, thapsigargin biosynthesis in T. garganica remains unknown. In order to decipher thapsigargin biosynthesis, deep transcript sequencing (454 and Illumina) of the T. garganica root was performed, and two terpene synthases (TgTPS1/2) were identified. Functional characterization of their encoded enzymes in a metabolically engineered yeast revealed that TgTPS1 synthesized δ-cadinene, whereas TgTPS2 produced ten distinct terpenoids. However, cultivation of the TgTPS2-expressing yeast in pH-maintained conditions (pH 6-7) yielded one major oxygenated sesquiterpenoid, suggesting that formation of multiple terpenoids was caused by acidity. The major terpene product from TgTPS2 was identified as 6β-hydroxygermacra-1(10),4-diene (kunzeaol) by mass-fragmentation pattern, retention index, the nature of its acid-induced degradation and NMR. Also, recombinant TgTPS2 efficiently catalysed the synthesis of kunzeaol in vitro from farnesyl diphosphate with a Km of 2.6 μM and a kcat of 0.03 s-1. The present paper is the first report of a kunzeaol synthase, and a mechanism for the transformation of kunzeaol into the thapsigargin backbone is proposed.
authors.affiliation: Department of Biological Sciences, University of Calgary, Calgary, Canada, T2N 1N4.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Pickel
authors.forename: Damian P
authors.initials: DP
authors.lastname: Drew
authors.forename: Tom
authors.initials: T
authors.lastname: Manczak
authors.forename: Corinna
authors.initials: C
authors.lastname: Weitzel
authors.forename: Henrik T
authors.initials: HT
authors.lastname: Simonsen
authors.forename: Dae-Kyun
authors.initials: DK
authors.lastname: Ro
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018744:DNA, Plant / Q000235:genetics; D008401:Gas Chromatography-Mass Spectrometry; D015870:Gene Expression; D017343:Genes, Plant; D007700:Kinetics; D008954:Models, Biological; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018517:Plant Roots / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism*; D053629:Thapsia / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D019284:Thapsigargin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D018744:DNA, Plant; D010940:Plant Proteins; D011994:Recombinant Proteins; D012717:Sesquiterpenes; C514849:kunzeaol; D019284:Thapsigargin; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase