Designed Water Capture in Terpene Synthase Catalysis

Sesquiterpene synthases catalyse cyclisations and rearrangements of farnesyl diphosphate to produce a diverse array of sesquiterpenes generated by depronotation and/or water capture. However, the precise mechanisms and dynamics controlling the fate of the final carbocationic intermediate are not well understood. In our previous study, we engineered water capture in selina-4(15),7(11)-diene synthase (SpSdS) to produce selin-7(11)-en-4-ol as a major product at pH 6.0 by point mutation (G305E) in the K_helix region. To develop a more generalised protocol for this functional switch in sesquiterpene synthases, we identified and characterised a novel selina-3,7(11)-diene synthase (AsSdS) from Actinacidiphila soli through multiple sequence alignments which naturally contains glutamate at position 305 (E305). Through site-directed mutagenesis, creating variant G221T, we were able to instigate water capture in AsSdS to produce selin-7(11)-en-4-ol. Our findings identified two crucial regions in the active site pocket of selinadiene synthases: G/E305 in K_helix and T/G221 in H_helix, that have a reproducible effect on product outcome determination. We demonstrate that subtle, yet predictable changes to these residues impact the water capture as well as deprotonation capability of selinadiene synthases and this solvation aspect can be further exploited to engineer other terpene synthases to generate biocatalysts with unique product profiles for diverse applications.