title: Mechanistic studies of sesquiterpene cyclases based on their carbon isotope ratios at natural abundance.
abstract: During the process of terpene biosynthesis, C-C bond breaking and forming steps are subjected to kinetic carbon isotope effects, leading to distinct carbon isotopic signatures of the products. Accordingly, carbon isotopic signatures could be used to reveal the 'biosynthetic history' of the produced terpenoids. Five known sesquiterpene cyclases, regulating three different pathways, representing simple to complex biosynthetic sequences, were heterologously expressed and used for in vitro assays with farnesyl diphosphate as substrate. Compound specific isotope ratio mass spectrometry measurements of the enzyme substrate farnesyl diphosphate (FDP) and the products of all the five cyclases were performed. The calculated δ13C value for FDP, based on δ13C values and relative amounts of the products, was identical with its measured δ13C value, confirming the reliability of the approach and the precision of measurements. The different carbon isotope ratios of the products reflect the complexity of their structure and are correlated with the frequency of carbon-carbon bond forming and breaking steps on their individual biosynthetic pathways. Thus, the analysis of carbon isotopic signatures of terpenes at natural abundance can be used as a powerful tool in elucidation of associated biosynthetic mechanisms of terpene synthases and in future in vivo studies even without 'touching' the plant.
authors.affiliation: Max Planck Institute for Chemical Ecology, Beutenberg Campus, Hans-Knöll-Str. 8, 07745, Jena, Germany.
authors.forename: Wenhua
authors.initials: W
authors.lastname: Tan
authors.affiliation: Max Planck Institute for Chemical Ecology, Beutenberg Campus, Hans-Knöll-Str. 8, 07745, Jena, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Bartram
authors.affiliation: Max Planck Institute for Chemical Ecology, Beutenberg Campus, Hans-Knöll-Str. 8, 07745, Jena, Germany.
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Boland
mesh_terms: D053898:Biosynthetic Pathways; D002247:Carbon Isotopes / Q000378:metabolism*; D019755:Carbon-Carbon Lyases / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D015203:Reproducibility of Results; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D002247:Carbon Isotopes; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D019755:Carbon-Carbon Lyases; C051130:trichodiene synthetase
keywords: biosynthetic pathway; carbon isotope effect; carbon isotopic signatures; gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS); gas chromatography-mass spectrometry (GC-MS); sesquiterpene; terpene cyclase; δ13C value