Discovery and Mechanistic Investigation of a Bacteria-Derived Sesquiterpene Ether Synthase AceS Catalyzing Syn -Protoetherification

Abstract

To discharge the carbocation in the termination step of terpene synthase (TS)-catalyzed cyclization, in addition to proton elimination to form alkene products and water quenching to afford hydroxylated products, intramolecular hydroxy group-trapping to give terpene ethers represents a variation of the latter case. Among the limited known TSs that are able to directly convert the linear oligoprenyl diphosphates to terpene ethers, 1,8-cineole synthase is the only characterized enzyme that catalyzes syn-protoetherification of the double bond in the termination step. We herein report the discovery and mechanistic investigation of a bacteria-derived sesquiterpene synthase AceS, converting farnesyl diphosphate to acorenol ether (1) in a syn-protoetherification manner. The unique 5/6 spiro-fused acorane skeleton with an ether ring bridging C3 and C7 of 1 was elucidated based on NMR analysis and X-ray diffraction (XRD) of a cytochrome P450-modified derivative. Labeling experiments using our recently developed “deuterium-scanning” approach allowed us to characterize a 1,2-hydride shift from C10 to C11 and a hydride shift from C6 to C10, as well as the syn-stereochemistry of the protoetherification process during cyclization. Results obtained from density functional theory (DFT) calculations, protein structure-guided site-directed mutagenesis, isolation and structural characterization of the products generated by the mutants, and protein–ligand docking simulations suggested cyclization through bisabolyl-dunnienyl cations as the favored pathway, albeit the coexistence of a pathway through carotenyl cations is also possible. Y186 may play as a key residue to redirect water to trap the C7 carbocation and to promote the syn-protoetherification reaction.