Electrostatic effects on (di)terpene synthase product outcome

Abstract

Terpene synthases catalyze complex reactions, often forming multiple chiral centers in cyclized olefin products from acyclic allylic diphosphate precursors, yet have been suggested to exert little control over the actual reaction, instead largely serving as inert templates. However, recent results highlight stereoelectronic effects exerted by these enzymes. Perhaps not surprisingly, the pyrophosphate co-product released in the initiating and rate-limiting chemical step provides an obvious counter-ion that may drive carbocation migration towards itself. This is emphasized by the striking effects of a recently uncovered single residue switch for diterpene synthase product outcome, whereby substitution of hydroxyl residues for particular aliphatic residues has been shown to be sufficient to “short-circuit” complex cyclization and/or rearrangement reactions, with the converse change further found to be sufficient to increase reaction complexity. The mechanistic hypothesis for the observed effects is hydroxyl dipole stabilization of the specific carbocation formed by initial cyclization, enabling deprotonation of this early intermediate, whereas the lack of such stabilization (i.e. with an aliphatic side chain) leads to carbocation migration towards the pyrophosphate co-product, resulting in a more complex reaction. This is further consistent with the greater synergy exhibited between pyrophosphate and aza-analogs of late, relative to early, stage carbocation intermediates, and crystallographic analysis of the monoterpene cyclase bornyl diphosphate synthase wherein mechanistically non-relevant counter-ion pairing between aza-analogs of early stage carbocation intermediates and pyrophosphate is observed. Thus, (di)terpene synthases seem to mediate specific reaction outcomes, at least in part, by providing stereoelectronic effects to counteract those exerted by the pyrophosphate co-product.

Identification of amino acid networks governing catalysis in the closed complex of class I terpene synthases

“…(vi) This steering and cation-attracting role of R580-PP i leads to direct premature deprotonation in cations C-F. (vii) It further leads to direct or indirect premature deprotonation when the finetuned and electrostatically balanced timeline of cyclization and hydride shifts is perturbed. (viii) Instead, in the absence of counteracting amino acid-assisted electrostatic effects, steric restriction imposed by the active site and steering of R580-PP i provide for cyclization from cation A→E, which corroborates the template model and dominating kinetic instead of thermodynamic control (24,54). (ix) Due to proximity of the positively charged carbocation to R580-PP i and in agreement with docking cluster analyses, cations F and E comprise lower energy levels in TXS compared with cation C in relation to cation A.…”

“…S3B). This strongly supports the model that terpene synthases mainly dictate reaction outcome by sterically restricting their substrate and discrete reaction intermediates to a subset of possible productive conformations (template model) (22,24). Moreover, it demonstrates differing binding modes followed by nonnative cyclization characteristics for substrate analogs that hamper the correct prediction of important structure-function relationships in class I terpene synthases.…”

“…1). However, structural data and mechanistic considerations explaining this promiscuity on the molecular level are very limited (8,(21)(22)(23)(24)(25). The role of the protein scaffold has been widely thought to chaperone the cyclization cascade by merely conformational control (22,26).…”

“…Very recently, a hypothesis pointing to a possible involvement of electrostatic effects during the carbocation cascade of other terpene synthases has been proposed. These effects are thought to be mediated by the PP i anion coproduct that may be retained in the active site (21,24,(26)(27)(28)(29)(30)(31)(32).…”

Characterisation of a Recombinant Patchoulol Synthase Variant for Biocatalytic Production of Terpenes

“…Several reaction types such as ring-closures, long-range hydrid-shifts and Wagner-Meerwein rearrangements are part of nature's toolbox of terpene formation which is required to build up the diverse and often complex carbon backbones of sesquiterpenoids [4,12]. In these reactions, π-cation and hydrophobic interactions play an important role, whereas the role of the cleaved-off diphosphate is still unsolved [52,53]. However, the stability of the numerous energetic often similar, transition states and intermediates is crucial for the selectivity of which particular product is formed by the multi-product terpene synthase and either under thermodynamic or kinetic control, which implies a temperature dependence [54].…”