Crystal Structure and Catalytic Mechanism of Drimenol Synthase, a Bifunctional Terpene Cyclase-Phosphatase

Drimenol synthase from Aquimarina spongiae (AsDMS) is a highly unusual bifunctional sesquiterpene synthase that integrates two distinct, sequential isoprenoid processing activities within a single polypeptide chain. AsDMS catalyzes the class II cyclization of farnesyl diphosphate (FPP) to form drimenyl diphosphate, which then undergoes enzyme-catalyzed hydrolysis to yield drimenol, a bioactive sesquiterpene alcohol with antifungal and anticancer properties. Here, we report the X-ray crystal structures of AsDMS and its complex with a sesquiterpene thiol, which are the first of a terpene cyclase-phosphatase. The AsDMS structure exhibits a two-domain architecture consisting of a terpene cyclase β domain and a haloacid dehalogenase (HAD)-like phosphatase domain, with two distinct active sites located on opposite sides of the protein. Mechanistic studies show that dephosphorylation of the drimenyl diphosphate intermediate proceeds through stepwise hydrolysis, such that two equivalents of inorganic phosphate rather than inorganic pyrophosphate are co-products of the reaction sequence. When the AsDMS reaction is performed in H₂ ¹⁸O, ¹⁸O is not incorporated into drimenol, indicating that the hydroxyl oxygen of drimenol originates from the prenyl oxygen of FPP rather than bulk water. These results correct a mechanistic proposal previously advanced by another group. Surprisingly, AsDMS exhibits unprecedented substrate promiscuity, catalyzing the conversion of substrate mimic farnesyl-S-thiolodiphosphate into cyclic and linear sesquiterpene products. Structural and mechanistic insights gained from AsDMS expand the functional diversity of terpene biosynthetic enzymes and provide a foundation for engineering "designer cyclases" capable of generating new terpenoid products.