# Designed Water Capture in Terpene Synthase Catalysis

## TL;DR

Researchers identified key regions in a terpene synthase that control whether water is captured or a proton is removed during the final step of a chemical reaction, enabling the design of new biocatalysts.

## Contribution

The study identifies two active site regions in selinadiene synthases that reproducibly control product outcomes through water capture or deprotonation.

## Key findings

- Two active site residues (G/E305 in Khelix and T/G221 in Hhelix) influence the final catalytic step in selinadiene synthases.
- Modifying these residues allows predictable switching between water capture and deprotonation to alter product profiles.
- The findings provide a generalizable strategy for engineering terpene synthases to create new biocatalysts.

## Abstract

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 Khelix 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 Khelix and T/G221 in Hhelix, 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.

Engineering of water capture in terpene synthases remains very challenging. We identified two regions in the selinadiene synthase active site pocket that can influence deprotonation or water capture in the final catalytic step and demonstrate a designed‐in switch in product outcome. These insights can be used for engineering other sesquiterpene synthases to generate new biocatalysts.© 2026 WILEY‐VCH GmbH