A diterpene synthase from the clary sage Salvia sclarea catalyzes the cyclization of geranylgeranyl diphosphate to (8R)-hydroxy-copalyl diphosphate |
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| Institution: | 1. Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, MO 63132, USA;2. Leibniz-Institut für Pflanzenbiochemie, Weinberg 3, D-06120 Halle (Saale), Germany;1. School of Life Sciences, Beijing University of Chinese Medicine, China;2. Beijing Key Laboratory of Analysis and Evaluation on Chinese Medicine, Beijing Institute for Drug Control, China;1. Department of Biochemistry and Biophysics, School of Basic Medical Sciences, Peking University, Beijing, 100191, China;2. Department of Chemistry, Capital Normal University, Beijing, 100048, China;3. National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China;1. Department of Chemical, Biochemical and Environmental Engineering, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, United States;2. Department of Food Science and Engineering, Jinan University, 601 West Huangpu Road, Guangzhou 510632, China;1. Urologic Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland;2. Developmental Therapeutics Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland;3. Departments of Urology and Cancer Genetics, Roswell Park Cancer Institute, Buffalo;4. Department of Urology, State University of New York, Upstate Medical University, Syracuse, New York;3. Departments of Biochemistry and Molecular Biology, East Lansing, Michigan 48824;4. Pharmacology and Toxicology, East Lansing, Michigan 48824;5. Michigan State University, East Lansing, Michigan 48824 |
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| Abstract: | The bicyclic diterpene (?)-sclareol is accumulated in glandular trichomes in Salvia sclarea (Schmiderer et al., 2008), and is a major terpenoid component of this plant species. It is used as the starting material for Ambrox synthesis, a synthetic ambergris analog used in the flavor and fragrance industry. In order to investigate the formation of sclareol, cDNA prepared from secretory cells of glandular trichomes from S. sclarea inflorescence were randomly sequenced. A putative copalyl diphosphate synthase encoding EST, SscTPS1, was functionally expressed in Escherichia coli. Whereas reaction of geranylgeranyl diphosphate with the putative copalyl diphosphate synthase followed by hydrolysis with alkaline phosphatase yielded a diastereomeric mixture of (13R)- and (13S)-manoyl oxide, HCl hydrolysis yielded (?)-sclareol (1) and 13-epi-sclareol as products. The product of the reaction of SscTPS1 with geranylgeranyl diphosphate was subjected to analysis by LC-negative ion ESI-MS/MS without prior hydrolysis. EPI scans were consistent with copalyl diphosphate to which 18 mass units had added (m/z 467 M+H]?). The enzymatic reaction was also carried out in the presence of 60% H218O. LC-negative ion ESI-MS/MS analysis established an additional reaction product consistent with the incorporation of 18O. Incubation in the presence of 60% 2H2O resulted in the incorporation of one deuterium atom. These results suggest water capture of the carbocation intermediate, which is known to occur in reactions catalyzed by monoterpene synthases, but has been described only several times for diterpene synthases. |
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