title: A diterpene synthase from the clary sage Salvia sclarea catalyzes the cyclization of geranylgeranyl diphosphate to (8R)-hydroxy-copalyl diphosphate.
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% H2(18)O. LC-negative ion ESI-MS/MS analysis established an additional reaction product consistent with the incorporation of (18)O. Incubation in the presence of 60% (2)H2O 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.
authors.affiliation: Donald Danforth Plant Science Center, St. Louis, MO 63132, USA.
authors.forename: Nils
authors.initials: N
authors.lastname: Günnewich
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Higashi
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Feng
authors.forename: Kum-Boo
authors.initials: KB
authors.lastname: Choi
authors.forename: Jürgen
authors.initials: J
authors.lastname: Schmidt
authors.forename: Toni M
authors.initials: TM
authors.lastname: Kutchan
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D055162:Biocatalysis; D003500:Cyclization; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010755:Organophosphates / Q000737:chemistry / Q000378:metabolism*; D011106:Polyisoprenyl Phosphates / Q000737:chemistry / Q000378:metabolism*; D027544:Salvia / Q000201:enzymology*; D012639:Seeds / Q000201:enzymology
chemical_list: C586028:(8R)-hydroxy-copalyl diphosphate; D010755:Organophosphates; D011106:Polyisoprenyl Phosphates; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase; C002963:geranylgeranyl pyrophosphate