Structure of Bifunctional Variediene Synthase Yields Unique Insight on Biosynthetic Diterpene Assembly and Cyclization.

An unusual family of bifunctional terpene synthases has been discovered in which both catalytic domains - a prenyltransferase and a cyclase - are connected by a long, flexible linker. These enzymes are unique to fungi and catalyze the first committed steps in the biosynthesis of complex terpenoid natural products: the prenyltransferase assembles 5-carbon precursors to form C geranylgeranyl diphosphate (GGPP), and the cyclase converts GGPP into a polycyclic hydrocarbon product. Weak domain-domain interactions as well as linker flexibility render these enzymes refractory to crystallization and challenge their visualization by cryo-EM. Despite these challenges, we now present the first experimentally-determined structure of a massive, 495-kD bifunctional terpene synthase revealing the assembly of all catalytic domains. The cryo-EM structure of variediene synthase from (EvVS) exhibits a bollard-like architecture, consisting of a hexameric prenyltransferase core sandwiched between two triads of cyclase domains. Although prenyltransferase and cyclase active sites are relatively close together, enzymological measurements indicate that GGPP is not channeled from one to the other. Surprisingly, however, the individual cyclase domain from another bifunctional diterpene synthase, fusicoccadiene synthase from , preferentially receives GGPP from the EvVS prenyltransferase in substrate competition experiments. Our previous studies of fusicoccadiene synthase suggest that GGPP channeling occurs through transient binding of cyclase domains to the sides of the prenyltransferase oligomer. The bollard-like architecture of EvVS leaves the sides of the prenyltransferase oligomer open and accessible, suggesting that a non-native cyclase could bind to the sides of the prenyltransferase oligomer to achieve GGPP channeling.