A Mechanism for Evolving Novel Plant Sesquiterpene Synthase Function

Plant sesquiterpene synthases, a subset of the terpene synthase superfamily, are a mechanistically diverse family of enzymes capable of synthesizing hundreds of complex compounds with high regio‐ and stereospecificity and are of biological importance due to their role in plant defense mechanisms. In the current report we describe a large‐scale, high‐resolution phylogenetic analysis of ∼200 plant sesquiterpene synthases integrated with structural and experimental data that address these issues. We observe that all sequences that cluster together on the phylogenetic tree into well‐defined groups share at least the first reaction in the catalytic mechanism subsequent to the initial ionization step and many share steps beyond this down to proton transfers between the enzyme and substrate. Most significant is the previously unreported high conservation of an Asp‐Tyr‐Asp triad. Due to its high conservation, patterns in the phylogenetic tree as well as experimental and modeling results, we suggest that this Asp‐Tyr‐Asp triad is an important functional element responsible for many proton transfers to and from the substrate and intermediates along the plant sesquiterpene synthase catalytic cycle and whose position can be tuned by residues outside the active site that can lead to the evolution of novel enzyme function.