Quantum chemical calculations on cyclization mechanisms for several sesquiterpene families proposed to be closely related to each other in a biogenic sense (the bisabolene, curcumene, acoradiene, zizaene (zizaene, isozizaene, epi-zizaene, and epi-isozizaene), cedrene (alpha/beta-cedrenes and 7-epi-alpha/beta-cedrenes), duprezianene, and sesquithuriferol families) are described. On the basis of the results of these calculations, we suggest that the conformation of the bisabolyl cation attainable in an enzyme active site is a primary determinant of the structure and relative stereochemistry of the sesquiterpenes formed. We also suggest that substantial conformational changes of initially formed conformers of the bisabolyl cation are necessary in order to form zizaene and epi-cedrene. Given that the productive conformation of the bisabolyl cation does not necessarily reflect the original orientation of farnesyl diphosphate bound in the corresponding enzyme active site, we conclude that folding of farnesyl diphosphate alone does not always dictate the structure and relative stereochemistry of cyclization products. In addition, the potential roles of dynamic matching in determining product distributions and enzyme-promoted formation of secondary carbocations are discussed.

Sesterterpenoids are a rare terpene class harboring untapped chemodiversity and bioactivities. Their structural diversity originates primarily from the scaffold-generating sesterterpene synthases (STSs). In fungi, all six known STSs are bifunctional, containing C-terminal trans-prenyltransferase (PT) and N-terminal terpene synthase (TPS) domains. In plants, two colocalized PT and TPS gene pairs from Arabidopsis thaliana were recently reported to synthesize sesterterpenes. However, the landscape of PT and TPS genes in plant genomes is unclear. Here, using a customized algorithm for systematically searching plant genomes, we reveal a suite of physically colocalized pairs of PT and TPS genes for the biosynthesis of a large sesterterpene repertoire in the wider Brassicaceae. Transient expression of seven TPSs from A. thaliana, Capsella rubella, and Brassica oleracea in Nicotiana benthamiana yielded fungal-type sesterterpenes with tri-, tetra-, and pentacyclic scaffolds, and notably (−)-ent-quiannulatene, an enantiomer of the fungal metabolite (+)-quiannulatene. Protein and structural modeling analysis identified an amino acid site implicated in structural diversification. Mutation of this site in one STS (AtTPS19) resulted in premature termination of carbocation intermediates and accumulation of bi-, tri-, and tetracyclic sesterterpenes, revealing the cyclization path for the pentacyclic sesterterpene (−)-retigeranin B. These structural and mechanistic insights, together with phylogenetic analysis, suggest convergent evolution of plant and fungal STSs, and also indicate that the colocalized PT-TPS gene pairs in the Brassicaceae may have originated from a common ancestral gene pair present before speciation. Our findings further provide opportunities for rapid discovery and production of sesterterpenes through metabolic and protein engineering.sesterterpene biosynthesis | plant natural products | cyclization mechanism | convergent evolution | Brassicaceae S esterterpenoids are a largely unexplored class of terpenes with only around 1,000 members isolated from nature so far, representing a mere <2% of the reported terpene family members (>70,000). These compounds are structurally diverse and have a wide spectrum of biological activities, ranging from antiinflammatory, anticancer, cytotoxic, and antimicrobial bioactivities to phytotoxicity and plant defense (1). Recent work on sesterterpenoids has suggested that this terpene class could be an important new source of anticancer drugs (2, 3). The majority of sesterterpenoids characterized to date are from marine sponges and terrestrial fungi, with only ∼60-70 being of plant origin. Many of these plant sesterterpenes were isolated from the mint family (Lamiaceae) and have been implicated in plant defense (4-7). Although large transcriptome datasets for the mint family have been generated (8), very limited genome sequences for the Lamiaceae are currently available (9, 10).Like other classes of terpenes, the structural diversity of sesterterpenes largely originates from the...

Selectivity in chemical reactions that form complex molecular architectures from simpler precursors is usually rationalized by comparing competing transition-state structures that lead to different possible products. Herein we describe a system for which a single transition-state structure leads to the formation of many isomeric products via pathways that feature multiple sequential bifurcations. The reaction network described connects the pimar-15-en-8-yl cation to miltiradiene, a tricyclic diterpene natural product, and isomers via cyclizations and/or rearrangements. The results suggest that the selectivity of the reaction is controlled by (post-transition-state) dynamic effects, that is, how the carbocation structure changes in response to the distribution of energy in its vibrational modes. The inherent dynamical effects revealed herein (characterized through quasiclassical direct dynamics calculations using density functional theory) have implications not only for the general principles of selectivity prediction in systems with complex potential energy surfaces, but also for the mechanisms of terpene synthase enzymes and their evolution. These findings redefine the challenges faced by nature in controlling the biosynthesis of complex natural products.

Terpenes comprise a class of natural products that includes molecules with thousands of distinct structurally and stereochemically complex molecular architectures. The core hydrocarbon frameworks of these molecules are constructed via carbocation rearrangements promoted by terpene synthase (cyclase) enzymes. Although many mechanistic details for such reactions have been uncovered, the factors that control which carbocation intermediates and transition-state structures form are not well understood. Here we show that rearrangement pathways that pass through particular transition-state structures can bifurcate after the transition state. The resulting pathways lead to terpenes with distinctly different skeletons from each other. Although these types of bifurcating pathways have been described previously for some small molecules, the possibility that they may have an important role in the production of complex molecules in nature has, to our knowledge, not previously been considered.

Secondary metabolites are major constituents of plant defense against herbivore attack. Relatively little is known about the cell type-specific formation and antiherbivore activities of secondary compounds in roots despite the substantial impact of root herbivory on plant performance and fitness. Here, we describe the constitutive formation of semivolatile diterpenes called rhizathalenes by the class I terpene synthase (TPS) 08 in roots of Arabidopsis thaliana. The primary enzymatic product of TPS08, rhizathalene A, which is produced from the substrate all-trans geranylgeranyl diphosphate, represents a so far unidentified class of tricyclic diterpene carbon skeletons with an unusual tricyclic spiro-hydrindane structure. Protein targeting and administration of stable isotope precursors indicate that rhizathalenes are biosynthesized in root leucoplasts. TPS08 expression is largely localized to the root stele, suggesting a centric and gradual release of its diterpene products into the peripheral root cell layers. We demonstrate that roots of Arabidopsis tps08 mutant plants, grown aeroponically and in potting substrate, are more susceptible to herbivory by the opportunistic root herbivore fungus gnat (Bradysia spp) and suffer substantial removal of peripheral tissue at larval feeding sites. Our work provides evidence for the in vivo role of semivolatile diterpene metabolites as local antifeedants in belowground direct defense against root-feeding insects.