How a 10-epi-cubebol Synthase Avoids Premature Reaction Quenching to Form a Tricyclic Product at High Purity

Terpenes are the largest class of natural products, and are attractive targets in the fuel, fragrance, pharmaceutical, and flavour industries. Harvesting terpenes from natural sources is environmentally intensive, and often gives low yields and purities, requiring further downstream processing. Engineered terpene synthases (TSs) offer a solution to these problems, but the low sequence identity and high promiscuity amongst TSs are major challenges for targeted engineering. Rational design of TSs requires identification of key structural and chemical motifs that steer product outcomes. Producing the sesquiterpenoid10-epi-cubebol from farnesyl pyrophosphate (FPP) requires many steps, and some of Nature’s most difficult chemistry. 10-epi-cubebol synthase from Sorangium cellulosum (ScCubS) guides a highly reactive carbocationic substrate through this pathway, preventing early quenching, and ensuring correct stereochemistry at every stage. The cyclisations carried out by ScCubS potentially represent significant evolutionary expansions in the chemical space accessible by TSs. Here we present the high-resolution crystal structure of ScCubS in complex with both a
trinuclear magnesium cluster and pyrophosphate. Computational modelling, experiment, and bioinformatic analysis identified residues important in steering the reaction chemistry. We show that S206 is crucial in 10-epi-cubebol synthesis by enlisting the nearby F211 to shape the active site contour and prevent the formation of early escape cadalane products. We also show that N327 and F104 control the distribution between several early-stage cations andwhether the final product is derived from the germacrane, cadalane, or cubebane hydrocarbon scaffold. Using these insights, we re-engineered ScCubS so that its main product
was germacradien-4-ol, which derives from the germacrane, rather than the cubebane, scaffold. Our work emphasises that mechanistic understanding of cation stabilisation in TSs can be used to guide catalytic outcomes.