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Terpene cyclases catalyze the synthesis of cyclic terpenes with 10-, 15-, and 20-carbon acyclic isoprenoid diphosphates as substrates. Plants have been a source of these natural products by providing a homologous set of terpene synthases. The crystal structures of 5-epi-aristolochene synthase, a sesquiterpene cyclase from tobacco, alone and complexed separately with two farnesyl diphosphate analogs were analyzed. These structures reveal an unexpected enzymatic mechanism for the synthesis of the bicyclic product, 5-epi-aristolochene, and provide a basis for understanding the stereochemical selectivity displayed by other cyclases in the biosynthesis of pharmacologically important cyclic terpenes. As such, these structures provide templates for the engineering of novel terpene cyclases.
Terpene cyclases control the synthesis of cyclic terpenoids including flavors and fragrances such as menthol and camphor, plant defense chemicals like capsidiol and lubimin ( 1), and more common compounds like steroids and lipid-soluble vitamins. Several cyclic terpenoids have pharmacological activity; for example, limonene can inhibit tumorigenesis induced in mice by particular carcinogens (2), and the diterpenoid taxol has antitumor activity (3). Numerous terpene cyclases from plant and microbial sources have been characterized (4, 5). Although the plant cyclases exhibit a significant degree of similarity in amino acid sequence, very little similarity is observed between the bacterial, fungal, and plant terpene cyclases (6). These soluble enzymes convert the acyclic isoprenoid diphosphates geranyl diphosphate (GPP, 10 carbon), farnesyl diphosphate (FPP, 15 carbon), and geranylgeranyl diphosphate (GGPP, 20 carbon) into cyclic monoterpenes, sesquiterpenes, and diterpenes, respectively. In most cases, loss of diphosphate (pyrophosphate) from the enzyme-bound acyclic substrate results in an allylic carbocation that electrophilically attacks a double bond further down the terpene chain to effect the first ring closure. Additional rearrangements involving transient carbocations can include proton abstractions, hydride and alkyl migrations, and additional electrophilic attacks.
TEAS (tobacco 5-epi-aristolochene synthase) (7) from Nicotiana tabacum converts farnesyl diphosphate (FPP) to 5-epiaristolochene (Fig. 1) (8), a precursor of the antifungal phytoalexin capsidiol. TEAS shares 77% amino acid identity with Hyoscyamus muticus vetispiradiene synthase (HVS) (9). Vetispiradiene (Fig. 1) is a precursor to the phytoalexins solavetivone and lubimin. Both enzymes have similar reaction mechanisms (9); in fact, several TEAS-HVS chimeras produce mixtures of the natural reaction products 5-epi-aristolochene and vetispiradiene (10).
In order to better understand terpene cyclase mechanisms, we have determined...