Phylogeny-directed discovery and mutagenesis of a tricyclic gersemiane synthase

Eunicellane diterpenoids, characterized by a 6,10-bicyclic core, are prone to undergo further cyclization into gersemiane diterpenoids <i>via</i> acid- or epoxidation-mediated protonation. However, it remains unclear whether classical type I terpene synthases (TSs), phylogenetically related to reported eunicellane synthases, can catalyze this protonation reaction, a process typically driven by type II TSs. To address this question, we employed a phylogeny-directed discovery approach starting from the eunicellane synthase MicA. This led to the discovery of SsFitTS, a tricyclic gersemiane synthase capable of catalyzing the protonation of a natural eunicellane intermediate within its classical type I TS active pocket. Through mutagenesis and molecular docking studies, we elucidated the mechanistic basis for this type I TS's ability to mediate a typically type II-like reaction while retaining its canonical type I fold. Our study not only demonstrated that the phylogeny-based genome mining method can unveil cryptic TSs but also facilitated the decoding of the structural basis underlying the determination of terpene scaffolds.