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Rodney Croteau - One of the best experts on this subject based on the ideXlab platform.
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alternative termination chemistries utilized by monoterpene cyclases chimeric analysis of bornyl diphosphate 1 8 cineole and Sabinene synthases
Archives of Biochemistry and Biophysics, 2003Co-Authors: Reuben J Peters, Rodney CroteauAbstract:Monoterpene cyclization reactions are initiated by ionization and isomerization of geranyl diphosphate, and proceed, via cyclization of bound linalyl diphosphate, through a series of carbocation intermediates with ultimate termination of the multistep cascade by deprotonation or nucleophile capture. Three structurally and mechanistically related monoterpene cyclases from Salvia officinalis, (+)-Sabinene synthase (deprotonation to olefin), 1,8-cineole synthase (water capture), and (+)-bornyl diphosphate synthase (diphosphate capture), were employed to explore the structural determinants of these alternative termination chemistries. Results with chimeric recombinant enzymes, constructed by reciprocally substituting regions of Sabinene synthase with the corresponding sequences from bornyl diphosphate synthase or 1,8-cineole synthase, demonstrated that exchange of the C-terminal catalytic domain is sufficient to completely switch the resulting product profile. Exchange of smaller sequence elements identified a region of roughly 70 residues from 1,8-cineole synthase that, when substituted into Sabinene synthase, conferred the ability to produce 1,8-cineole. A similar strategy identified a small region of bornyl diphosphate synthase important in conducting the anti-Markovnikov addition to the bornane skeleton. Observations made with these chimeric monoterpene cyclases are discussed in the context of the recently determined crystal structure for bornyl diphosphate synthase.
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Monoterpene Biosynthesis in the Liverwort Conocephalum Conicum: Demonstration of Sabinene Synthase and Bornyl Diphosphate Synthase in Honour of Professor G. H. Neil Towers 75th Birthday
Phytochemistry, 1998Co-Authors: Klaus-peter Adam, Rodney CroteauAbstract:Abstract (−)-Sabinene is the major monoterpene produced by a European strain of the liverwort Conocephalum conicum. A cell-free extract from in vitro cultured plants catalysed the cyclization of geranyl diphosphate to Sabinene. The responsible monoterpene cyclase was partially purified and characterized as an operationally soluble enzyme of Mr 65 000, with a pH optimum at 7.5 and a requirement for a divalent metal ion as the only cofactor, with Mg2 preferred. The general properties of the Sabinene synthase from C. conicum resemble those of other monoterpene cyclases from gymnosperms and angiosperms. A North American strain of the liverwort produces (+)-bornyl acetate as the major monoterpene and it was demonstrated that bornane-type monoterpenes are derived from geranyl diphosphate in this liverwort, as in higher plants, by the action of bornyl diphosphate synthase.
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monoterpene synthases from common sage salvia officinalis cdna isolation characterization and functional expression of Sabinene synthase 1 8 cineole synthase and bornyl diphosphate synthase
Journal of Biological Chemistry, 1998Co-Authors: Mitchell L Wise, Thomas J Savage, Eva Katahira, Rodney CroteauAbstract:Abstract Common sage (Salvia officinalis) produces an extremely broad range of cyclic monoterpenes bearing diverse carbon skeletons, including members of thep-menthane (1,8-cineole), pinane (α- and β-pinene), thujane (isothujone), camphane (camphene), and bornane (camphor) families. An homology-based polymerase chain reaction cloning strategy was developed and used to isolate the cDNAs encoding three multiproduct monoterpene synthases from this species that were functionally expressed in Escherichia coli. The heterologously expressed synthases produce (+)-bornyl diphosphate, 1,8-cineole, and (+)-Sabinene, respectively, as their major products from geranyl diphosphate. The bornyl diphosphate synthase also produces significant amounts of (+)-α-pinene, (+)-camphene, and (±)-limonene. The 1,8-cineole synthase produces significant amounts of (+)- and (−)-α-pinene, (+)- and (−)-β-pinene, myrcene and (+)-Sabinene, and the (+)-Sabinene synthase produces significant quantities of γ-terpinene and terpinolene. All three enzymes appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence, consistent with the plastidial origin of monoterpenes in plants. Deduced sequence analysis and size exclusion chromatography indicate that the recombinant bornyl diphosphate synthase is a homodimer, whereas the other two recombinant enzymes are monomeric, consistent with the size and subunit architecture of their native enzyme counterparts. The distribution and stereochemistry of the products generated by the recombinant (+)-bornyl diphosphate synthase suggest that this enzyme might represent both (+)-bornyl diphosphate synthase and (+)-pinene synthase which were previously assumed to be distinct enzymes.
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Monoterpene Synthases from Common Sage (Salvia officinalis) cDNA ISOLATION, CHARACTERIZATION, AND FUNCTIONAL EXPRESSION OF (+)-Sabinene SYNTHASE, 1,8-CINEOLE SYNTHASE, AND (+)-BORNYL DIPHOSPHATE SYNTHASE
Journal of Biological Chemistry, 1998Co-Authors: Mitchell L Wise, Thomas J Savage, Eva Katahira, Rodney CroteauAbstract:Abstract Common sage (Salvia officinalis) produces an extremely broad range of cyclic monoterpenes bearing diverse carbon skeletons, including members of thep-menthane (1,8-cineole), pinane (α- and β-pinene), thujane (isothujone), camphane (camphene), and bornane (camphor) families. An homology-based polymerase chain reaction cloning strategy was developed and used to isolate the cDNAs encoding three multiproduct monoterpene synthases from this species that were functionally expressed in Escherichia coli. The heterologously expressed synthases produce (+)-bornyl diphosphate, 1,8-cineole, and (+)-Sabinene, respectively, as their major products from geranyl diphosphate. The bornyl diphosphate synthase also produces significant amounts of (+)-α-pinene, (+)-camphene, and (±)-limonene. The 1,8-cineole synthase produces significant amounts of (+)- and (−)-α-pinene, (+)- and (−)-β-pinene, myrcene and (+)-Sabinene, and the (+)-Sabinene synthase produces significant quantities of γ-terpinene and terpinolene. All three enzymes appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence, consistent with the plastidial origin of monoterpenes in plants. Deduced sequence analysis and size exclusion chromatography indicate that the recombinant bornyl diphosphate synthase is a homodimer, whereas the other two recombinant enzymes are monomeric, consistent with the size and subunit architecture of their native enzyme counterparts. The distribution and stereochemistry of the products generated by the recombinant (+)-bornyl diphosphate synthase suggest that this enzyme might represent both (+)-bornyl diphosphate synthase and (+)-pinene synthase which were previously assumed to be distinct enzymes.
Jorg Degenhardt - One of the best experts on this subject based on the ideXlab platform.
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stereochemical mechanism of two Sabinene hydrate synthases forming antipodal monoterpenes in thyme thymus vulgaris
Archives of Biochemistry and Biophysics, 2013Co-Authors: Sandra T Krause, Tobias G Kollner, Julia Asbach, Jorg DegenhardtAbstract:The essential oil of Thymus vulgaris consists of a complex blend of mono- and sesquiterpenes that provides the plant with its characteristic aromatic odor. Several chemotypes have been described for thyme. In this study, we identified two enzymes of the Sabinene hydrate chemotype which are responsible for the biosynthesis of its major monoterpene alcohols, (1S,2R,4S)-(Z)-Sabinene hydrate and (1S,2S,4R)-(E)-Sabinene hydrate. Both TPS6 and TPS7 are multiproduct enzymes that formed 16 monoterpenes and thus cover almost the whole monoterpene spectrum of the chemotype. Although the product spectra of both enzymes are similar, they form opposing enantiomers of their chiral products. Incubation of the enzymes with the potential reaction intermediates revealed that the stereospecificity of TPS6 and TPS7 is determined by the formation of the first intermediate, linalyl diphosphate. Since TPS6 and TPS7 shared an amino acid sequence identity of 85%, a mutagenesis study was employed to identify the amino acids that determine the stereoselectivity. One amino acid position had a major influence on the stereochemistry of the formed products. Based on comparative models of TPS6 and TPS7 protein structures with the GPP substrate docked in the active site pocket, the influence of this amino acid residue on the reaction mechanism is discussed.
Jörg Bohlmann - One of the best experts on this subject based on the ideXlab platform.
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the gymnosperm cytochrome p450 cyp750b1 catalyzes stereospecific monoterpene hydroxylation of Sabinene in thujone biosynthesis in western redcedar
Plant Physiology, 2015Co-Authors: Andreas Gesell, Markus Blaukopf, Lina Madilao, Macaire M S Yuen, John H Russell, Stephen G Withers, Jim Mattsson, Jörg BohlmannAbstract:Western redcedar (WRC; Thuja plicata) produces high amounts of oxygenated thujone monoterpenoids associated with resistance against herbivore feeding, particularly ungulate browsing. Thujones and other monoterpenoids accumulate in glandular structures in the foliage of WRC. Thujones are produced from (+)-Sabinene by sabinol and sabinone. Using metabolite analysis, enzyme assays with WRC tissue extracts, cloning, and functional characterization of cytochrome P450 monooxygenases, we established that trans-sabin-3-ol but not cis-sabin-3-ol is the intermediate in thujone biosynthesis in WRC. Based on transcriptome analysis, full-length complementary DNA cloning, and characterization of expressed P450 proteins, we identified CYP750B1 and CYP76AA25 as the enzymes that catalyze the hydroxylation of (+)-Sabinene to trans-sabin-3-ol. Gene-specific transcript analysis in contrasting WRC genotypes producing high and low amounts of monoterpenoids, including a glandless low-terpenoid clone, as well as assays for substrate specificity supported a biological role of CYP750B1 in α- and β-thujone biosynthesis. This P450 belongs to the apparently gymnosperm-specific CYP750 family and is, to our knowledge, the first member of this family to be functionally characterized. In contrast, CYP76AA25 has a broader substrate spectrum, also converting the sesquiterpene farnesene and the herbicide isoproturon, and its transcript profiles are not well correlated with thujone accumulation.
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The Gymnosperm Cytochrome P450 CYP750B1 Catalyzes Stereospecific Monoterpene Hydroxylation of (+)-Sabinene in Thujone Biosynthesis in Western Redcedar
Plant Physiology, 2015Co-Authors: Andreas Gesell, Markus Blaukopf, Lina Madilao, Macaire M S Yuen, John H Russell, Stephen G Withers, Jim Mattsson, Jörg BohlmannAbstract:Western redcedar (WRC; Thuja plicata) produces high amounts of oxygenated thujone monoterpenoids associated with resistance against herbivore feeding, particularly ungulate browsing. Thujones and other monoterpenoids accumulate in glandular structures in the foliage of WRC. Thujones are produced from (+)-Sabinene by sabinol and sabinone. Using metabolite analysis, enzyme assays with WRC tissue extracts, cloning, and functional characterization of cytochrome P450 monooxygenases, we established that trans-sabin-3-ol but not cis-sabin-3-ol is the intermediate in thujone biosynthesis in WRC. Based on transcriptome analysis, full-length complementary DNA cloning, and characterization of expressed P450 proteins, we identified CYP750B1 and CYP76AA25 as the enzymes that catalyze the hydroxylation of (+)-Sabinene to trans-sabin-3-ol. Gene-specific transcript analysis in contrasting WRC genotypes producing high and low amounts of monoterpenoids, including a glandless low-terpenoid clone, as well as assays for substrate specificity supported a biological role of CYP750B1 in α- and β-thujone biosynthesis. This P450 belongs to the apparently gymnosperm-specific CYP750 family and is, to our knowledge, the first member of this family to be functionally characterized. In contrast, CYP76AA25 has a broader substrate spectrum, also converting the sesquiterpene farnesene and the herbicide isoproturon, and its transcript profiles are not well correlated with thujone accumulation.
Christophe C Galopin - One of the best experts on this subject based on the ideXlab platform.
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a short and efficient synthesis of trans Sabinene hydrate
Tetrahedron Letters, 2001Co-Authors: Christophe C GalopinAbstract:Abstract A short synthesis of Sabinene hydrate is reported. It uses cheap starting materials and affordable reagents. The main product of the synthesis is trans -Sabinene hydrate.
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A short and efficient synthesis of (±)-trans-Sabinene hydrate
Tetrahedron Letters, 2001Co-Authors: Christophe C GalopinAbstract:Abstract A short synthesis of Sabinene hydrate is reported. It uses cheap starting materials and affordable reagents. The main product of the synthesis is trans -Sabinene hydrate.
Uzi Ravid - One of the best experts on this subject based on the ideXlab platform.
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enantiomeric composition of e and z Sabinene hydrate and their acetates in five origanum spp
Flavour and Fragrance Journal, 2005Co-Authors: Olga Larkov, Ezra Dunkelblum, Anat Zada, Efraim Lewinsohn, Lilia Freiman, Nativ Dudai, Uzi RavidAbstract:The enantiomers of (E)- and (Z)-Sabinene hydrate and of (E)- and (Z)-Sabinene hydrate acetate from extracts of Origanum ramonense Danin, O. dayi Post, O. majorana L., O. vulgare L. ssp. vulgare and O. syriacum L. ssp. syriacum were analysed by GC–MS with chiral and non-chiral capillary columns. The order of elution, the enantiomeric ratios and relative percentages of the four pairs of enantiomers were determined. The (1S)-enantiomers of (E)-Sabinene hydrate and (E)-Sabinene hydrate acetate were predominant in O. dayi, whereas in the other Origanum spp. the (1R)-enantiomers were predominant. (Z)-Sabinene hydrate acetate was not detected in O. syriacum, while the (1R)-enantiomer was present in an optically pure form in O. ramonense, O. majorana and O. vulgare; in O. dayi the enantiomeric purity was 97%. The enantiomeric distributions of (E)- and (Z)-Sabinene hydrate and their acetates were determined for the first time in Origanum spp. Copyright © 2004 John Wiley & Sons, Ltd.
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Enantiomeric composition of (E)‐ and (Z)‐Sabinene hydrate and their acetates in five Origanum spp.
Flavour and Fragrance Journal, 2005Co-Authors: Olga Larkov, Ezra Dunkelblum, Anat Zada, Efraim Lewinsohn, Lilia Freiman, Nativ Dudai, Uzi RavidAbstract:The enantiomers of (E)- and (Z)-Sabinene hydrate and of (E)- and (Z)-Sabinene hydrate acetate from extracts of Origanum ramonense Danin, O. dayi Post, O. majorana L., O. vulgare L. ssp. vulgare and O. syriacum L. ssp. syriacum were analysed by GC–MS with chiral and non-chiral capillary columns. The order of elution, the enantiomeric ratios and relative percentages of the four pairs of enantiomers were determined. The (1S)-enantiomers of (E)-Sabinene hydrate and (E)-Sabinene hydrate acetate were predominant in O. dayi, whereas in the other Origanum spp. the (1R)-enantiomers were predominant. (Z)-Sabinene hydrate acetate was not detected in O. syriacum, while the (1R)-enantiomer was present in an optically pure form in O. ramonense, O. majorana and O. vulgare; in O. dayi the enantiomeric purity was 97%. The enantiomeric distributions of (E)- and (Z)-Sabinene hydrate and their acetates were determined for the first time in Origanum spp. Copyright © 2004 John Wiley & Sons, Ltd.
