Substrate geometry controls the cyclization cascade in multiproduct terpene synthases from Zea mays.

Multiproduct terpene synthases TPS4-B73 and TPS5-Delprim from maize (Zea mays) catalyze the conversion of farnesyl diphosphate (FDP) and geranyl diphosphate (GDP) into a complex mixture of sesquiterpenes and monoterpenes, respectively. Various isotopic and geometric isomers of natural substrates like (2Z)-[2-(2)H]- and [2,4,4,9,9,9-(2)H6]-(GDP) and (2Z,6E)-[2-(2)H]- and [2,4,4,13,13,13-(2)H6]-(FDP) were synthesized analogous to presumptive reaction intermediates. On incubation with labeled (2Z) substrates, TPS4 and TPS5 showed much lower kinetic isotope effects than the labeled (2E) substrates. Interestingly, the products arising from the deuterated (2Z)-precursors revealed a distinct preference for cyclic products and exhibited an enhanced turnover on comparison with natural (2E)-substrates. This increase in the efficiency due to (2Z) configuration emphasizes the rate limiting effect of the initial (2E) → (2Z) isomerization step in the reaction cascade of the multiproduct terpene synthases. Apart from turnover advantages, these results suggest that substrate geometry can be used as a tool to optimize the biosynthetic reaction cascade towards valuable cyclic terpenoids.

Inhibition of a multiproduct terpene synthase from Medicago truncatula by 3-bromoprenyl diphosphates.

The multiproduct sesquiterpene synthase MtTPS5 from Medicago truncatula catalyzes the conversion of farnesyl diphosphate (FDP) into a complex mixture of 27 terpenoids. 3-Bromo substrate analogues of geranyl diphosphate (3-BrGDP) and farnesyl diphosphate (3-BrFDP) were evaluated as substrates of MTPS5 enzyme. Kinetic studies demonstrated that these compounds were highly potent competitive inhibitors of the MtTPS5 enzyme with fast binding and slow reversibility. Since there is a lack of knowledge about the crystal structure of multiproduct terpene synthases, these molecules might be ideal candidates for obtaining a co-crystal structure with multiproduct terpene synthases. Due to the structural and mechanistic similarity between various terpene synthases we expect these 3-bromo isoprenoids to be ideal probes for crystal structure studies.

Isotope sensitive branching and kinetic isotope effects to analyse multiproduct terpenoid synthases from Zea mays.

Multiproduct terpene synthases TPS4-B73 and TPS5-Delprim from Zea mays exhibit isotopically sensitive branching in the formation of mono- and sesquiterpene volatiles. The impact of the kinetic isotope effects and the stabilization of the reactive intermediates by hyperconjugation along with the shift of products from alkenes to alcohols are discussed.

Two pockets in the active site of maize sesquiterpene synthase TPS4 carry out sequential parts of the reaction scheme resulting in multiple products.

One of the most interesting features of terpene synthases is their ability to form multiple products with different carbon skeletons from a single prenyl diphosphate substrate. The maize sesquiterpene synthase TPS4, for example, produces a mixture of 14 different olefinic sesquiterpenes. To understand the complex TPS4 reaction mechanism, we modeled the active site cavity and conducted docking simulations with the substrate farnesyl diphosphate, several predicted carbocation intermediates, and the final reaction products. The model suggests that discrete steps of the reaction sequence are controlled by two different active site pockets, with the conformational change of the bisabolyl cation intermediate causing a shift from one pocket to the other. Site-directed mutagenesis and measurements of mutant activity in the presence of (E,E)- and (Z,E)-farnesyl diphosphate as substrates were employed to test this model. Amino acid alterations in pocket I indicated that early steps of the catalytic process up to the formation of the monocyclic bisabolyl cation are probably localized in this compartment. Mutations in pocket II primarily inhibited the formation of bicylic compounds, suggesting that secondary cyclizations of the bisabolyl cation are catalyzed in pocket II.

The nonmevalonate pathway supports both monoterpene and sesquiterpene formation in snapdragon flowers.

Terpenoids, the largest class of plant secondary metabolites, play essential roles in both plant and human life. In higher plants, the five-carbon building blocks of all terpenoids, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate, are derived from two independent pathways localized in different cellular compartments. The methylerythritol phosphate (MEP or nonmevalonate) pathway, localized in the plastids, is thought to provide IPP and dimethylallyl diphosphate for hemiterpene, monoterpene, and diterpene biosynthesis, whereas the cytosol-localized mevalonate pathway provides C5 units for sesquiterpene biosynthesis. Stable isotope-labeled, pathway-specific precursors (1-deoxy-[5,5-2H2]-D-xylulose and [2,2-2H2]-mevalolactone) were supplied to cut snapdragon flowers, which emit both monoterpenes and the sesquiterpene, nerolidol. We show that only one of the two pathways, the plastid-localized MEP pathway, is active in the formation of volatile terpenes. The MEP pathway provides IPP precursors for both plastidial monoterpene and cytosolic sesquiterpene biosynthesis in the epidermis of snapdragon petals. The trafficking of IPP occurs unidirectionally from the plastids to cytosol. The MEP pathway operates in a rhythmic manner controlled by the circadian clock, which determines the rhythmicity of terpenoid emission.

Are iridoids in leaf beetle larvae synthesized de novo or derived from plant precursors? A methodological approach.

Iridoids, belonging to a group of cyclopentanoid monoterpenoids, are secreted by many species of leaf beetles as a defense against predators. Using chemically modified precursors of iridoid biosynthesis, it has been shown that some leaf beetle larvae can synthesize these iridoids de novo as well as sequester plant-produced molecules. Stable isotope techniques can provide useful methods for studying terpenoid biosynthesis without disturbing the natural conditions much. Two terpenoid biosynthesis pathways (mevalonic acid (MVA) pathway and methylerythritol-4-phosphate (MEP) pathway) may lead to different delta13C signatures of the products. Our results from natural abundance 13C and 13C-labelled iridoid precursors in Gastrophysa viridula and Phaedon cochleariae suggested that the two leaf beetle species use only de novo synthesis of their defensive iridoids. We observed that the isotope signature of the leaf-beetle-produced iridoids (via the MVA pathway) resembled that of the MEP-derived monoterpenoids from plants. Owing to this close similarity in the natural 13C abundances in the plant and insect compounds, a determination of iridoid-origin in leaf beetle secretion may only be possible by use of isotopically labelled compounds.