Investigations into the function of two plant sesquiterpene synthases: δ-cadinene synthase and (e)-β-farnesene synthase

Terpenoids represent the most structurally and stereochemically diverse family of natural products with more than 55,000 terpenoid structures discovered to date from all life forms. Sesquiterpenes are a class of the terpenoid family, and their formation from farnesyl diphosphate is catalyzed by sesquiterpene synthases. This project focuses on trying to decipher the reaction mechanisms of two sesquiterpene synthases, 6-cadinene synthase from Gossypium arboreum and (2<r)- 3-farnesene synthase from Mentha x piperita and to provide a method for the generation of unnatural terpenes with potential commercial applications in both the pharmaceutical and agrochemical industries. Modifications by substitution of residues around the active site of 6-cadinene synthase did not lead to any functional divergence, indicating an unusual structural component that determines the product specificity of this enzyme. Domain-swapping experiments based on phylogenetic information suggested that the subdomain encoded by exon 4 is most likely the key structure element controlling the product specificity of this enzyme. Manipulation of the active site volume of (2T)-p-farnesene synthase by site-directed mutagenesis revealed a rigid active site cavity that is precisely defined for generating mainly acyclic products. The active site hybrid constructed by replacing the active surface of (&pound;)-p-farnesene synthase with the corresponding part from 6-cadinene synthase lost activity, suggesting the catalytic specificity of this enzyme is modulated at a distance by residues surrounding the active site, which may have a huge influence on the active site volume. Mechanistic studies utilizing a substrate analogue revealed a new reaction mechanism for (&pound;)-P-farnesene synthase. Functional approaches to explore the N-termini region of 6-cadinene synthase and (&pound;)-p-farnesene synthase provided direct evidence that suggested dual roles for this region

Probing the mechanism of 1,4-conjugate elimination reactions catalyzed by terpene synthases

The reaction mechanisms of (E)-β-farnesene synthase (EBFS) and isoprene synthase (ISPS), enzymes that catalyze a formal regiospecific 1,4-conjugate elimination of hydrogen diphosphate from (E,E)-farnesyl and dimethylallyl diphosphate (FDP and DMADP) to generate the semiochemicals (E)-β-farnesene and isoprene, respectively, were probed with substrate analogs and kinetic measurements. The results support stepwise reaction mechanisms through analogous enzyme-bound allylic cationic intermediates. For EBFS, we demonstrate that the elimination reaction can proceed via the enzyme-bound intermediate trans-nerolidyl diphosphate, while for ISPS the intermediacy of 2-methylbut-3-enyl 2-diphosphate can be inferred from the product outcome when deuterated DMADPs are used as substrates. Possible implications derived from the mechanistic details of the EBFS-catalyzed reaction for the evolution of sesquiterpene synthases are discussed

A 1,6-ring closure mechanism for (+)-δ-cadinene synthase?

Recombinant (+)-δ-cadinene synthase (DCS) from Gossypium arboreum catalyzes the metal-dependent cyclization of (E,E)-farnesyl diphosphate (FDP) to the cadinane sesquiterpene δ-cadinene, the parent hydrocarbon of cotton phytoalexins such as gossypol. In contrast to some other sesquiterpene cyclases, DCS carries out this transformation with >98% fidelity but, as a consequence, leaves no mechanistic traces of its mode of action. The formation of (+)-δ-cadinene has been shown to occur via the enzyme-bound intermediate (3R)-nerolidyl diphosphate (NDP), which in turn has been postulated to be converted to cis-germacradienyl cation after a 1,10-cyclization. A subsequent 1,3-hydride shift would then relocate the carbocation within the transient macrocycle to expedite a second cyclization that yields the cadinenyl cation with the correct cis stereochemistry found in (+)-δ-cadinene. An elegant 1,10-mechanistic pathway that avoids the formation of (3R)-NDP has also been suggested. In this alternative scenario, the final cadinenyl cation is proposed to be formed through the intermediacy of trans, trans-germacradienyl cation and germacrene D. In addition, an alternative 1,6-ring closure mechanism via the bisabolyl cation has previously been envisioned. We report here a detailed investigation of the catalytic mechanism of DCS using a variety of mechanistic probes including, among others, deuterated and fluorinated FDPs. Farnesyl diphosphate analogues with fluorine at C2 and C10 acted as inhibitors of DCS, but intriguingly, after prolonged overnight incubations, they yielded 2F-germacrene(s) and a 10F-humulene, respectively. The observed 1,10-, and to a lesser extent, 1,11-cyclization activity of DCS with these fluorinated substrates is consistent with the postulated macrocyclization mechanism(s) en route to (+)-δ-cadinene. On the other hand, mechanistic results from incubations of DCS with 6F-FPP, (2Z,6E)-FDP, neryl diphosphate, 6,7-dihydro-FDP, and NDP seem to be in better agreement with the potential involvement of the alternative biosynthetic 1,6-ring closure pathway. In particular, the strong inhibition of DCS by 6F-FDP, coupled to the exclusive bisabolyl- and terpinyl-derived product profiles observed for the DCS-catalyzed turnover of (2Z,6E)-farnesyl and neryl diphosphates, suggested the intermediacy of α-bisabolyl cation. DCS incubations with enantiomerically pure [1-2H1](1R)-FDP revealed that the putative bisabolyl-derived 1,6-pathway proceeds through (3R)-nerolidyl diphosphate (NDP), is consistent with previous deuterium-labeling studies, and accounts for the cis stereochemistry characteristic of cadinenyl-derived sesquiterpenes. While the results reported here do not unambiguously rule in favor of 1,6- or 1,10-cyclization, they demonstrate the mechanistic versatility inherent to DCS and highlight the possible existence of multiple mechanistic pathways

Crystal structure of (+)-δ-cadinene synthase from Gossypium arboreum and evolutionary divergence of metal binding motifs for catalysis

(+)-δ-Cadinene synthase (DCS) from Gossypium arboreum (tree cotton) is a sesquiterpene cyclase that catalyzes the cyclization of farnesyl diphosphate in the first committed step of the biosynthesis of gossypol, a phytoalexin that defends the plant from bacterial and fungal pathogens. Here, we report the X-ray crystal structure of unliganded DCS at 2.4 Å resolution and the structure of its complex with three putative Mg2+ ions and the substrate analogue inhibitor 2-fluorofarnesyl diphosphate (2F-FPP) at 2.75 Å resolution. These structures illuminate unusual features that accommodate the trinuclear metal cluster required for substrate binding and catalysis. Like other terpenoid cyclases, DCS contains a characteristic aspartate-rich D307DTYD311 motif on helix D that interacts with Mg2+A and Mg2+C. However, DCS appears to be unique among terpenoid cyclases in that it does not contain the “NSE/DTE” motif on helix H that specifically chelates Mg2+B, which is usually found as the signature sequence (N,D)D(L,I,V)X(S,T)XXXE (boldface indicates Mg2+B ligands). Instead, DCS contains a second aspartate-rich motif, D451DVAE455, that interacts with Mg2+B. In this regard, DCS is more similar to the isoprenoid chain elongation enzyme farnesyl diphosphate synthase, which also contains two aspartate-rich motifs, rather than the greater family of terpenoid cyclases. Nevertheless, the structure of the DCS−2F-FPP complex shows that the structure of the trinuclear magnesium cluster is generally similar to that of other terpenoid cyclases despite the alternative Mg2+B binding motif. Analyses of DCS mutants with alanine substitutions in the D307DTYD311 and D451DVAE455 segments reveal the contributions of these segments to catalysis

Probing the Mechanism of 1,4-Conjugate Elimination Reactions Catalyzed by Terpene Synthases

The reaction mechanisms of (<i>E</i>)-β-farnesene synthase (EBFS) and isoprene synthase (ISPS), enzymes that catalyze a formal regiospecific 1,4-conjugate elimination of hydrogen diphosphate from (<i>E</i>,<i>E</i>)-farnesyl and dimethylallyl diphosphate (FDP and DMADP) to generate the semiochemicals (<i>E</i>)-β-farnesene and isoprene, respectively, were probed with substrate analogs and kinetic measurements. The results support stepwise reaction mechanisms through analogous enzyme-bound allylic cationic intermediates. For EBFS, we demonstrate that the elimination reaction can proceed via the enzyme-bound intermediate <i>trans</i>-nerolidyl diphosphate, while for ISPS the intermediacy of 2-methylbut-3-enyl 2-diphosphate can be inferred from the product outcome when deuterated DMADPs are used as substrates. Possible implications derived from the mechanistic details of the EBFS-catalyzed reaction for the evolution of sesquiterpene synthases are discussed