A diterpene synthase from the clary sage Salvia sclarea catalyzes the cyclization of geranylgeranyl diphosphate to (8R)-hydroxy-copalyl diphosphate

• Published in: Phytochemistry (Oxford) 2013-07, Vol.91, p.93-99
• Main Authors: Günnewich, Nils, Higashi, Yasuhiro, Feng, Xiaohong, Choi, Kum-Boo, Schmidt, Jürgen, Kutchan, Toni M.
• Format: Article
• Language: English
• Subjects: alkaline phosphatase; Alkyl and Aryl Transferases - chemistry; Alkyl and Aryl Transferases - isolation & purification; Alkyl and Aryl Transferases - metabolism; Biocatalysis; Clary sage; complementary DNA; Cyclization; deuterium; Escherichia coli; expressed sequence tags; flavor; hydrochloric acid; hydrolysis; Hydroxy-copalyl diphosphate; Lamiaceae; Molecular Conformation; Molecular Sequence Data; monoterpenoids; odors; Organophosphates - chemistry; Organophosphates - metabolism; Polyisoprenyl Phosphates - chemistry; Polyisoprenyl Phosphates - metabolism; sage; Salvia - enzymology; Salvia sclarea; Sclareol; Seeds - enzymology; Terpene synthase; trichomes
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/j.phytochem.2012.07.019

The diterpene synthase SscTPS1 from glandular trichomes of Clary sage catalyzes cyclization of geranylgeranyl diphosphate by a mechanism that introduces oxygen to form (8R)-hydroxy-copalyl diphosphate, which can then be converted to sclareol and 13-epi-sclareol by acid hydrolysis. [Display omitted] ► A cDNA encoding a diterpene synthase from Salvia sclarea has been isolated. ► The recombinant enzyme converts GGPP to (8R)-hydroxy-copalyl diphosphate. ► Hydrolysis of the enzymic reaction with HCl yields (−)-sclareol and 13-epi-sclareol. 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.