Crystal Structure and Catalytic Mechanism of the <i>cis</i>‐Eunicellane Cyclase AriE

Eunicellane diterpenoids, with over 360 known examples characterized by a cis- or trans-6/10-bicyclic carbon skeleton, exhibit diverse biological activities. To date, five eunicellane cyclases (ECs) have been reported: cis-ECs (Bnd4, CpDTS2 and EcTPS1) and trans-ECs (AlbS and MicA), yet the structural basis and molecular mechanisms governing their distinct cis- or trans-cyclization modes remain unsolved. Here, we present the crystal structure of a cis-EC AriE from Amycolatopsis arida, which catalyzes geranylgeranyl diphosphate to form cis-6/10-fused bicyclic benditerpe-2,6,15-triene (1) in aridacin biosynthesis. Through surface entropy reduction, we obtained the 1.87 Å resolution structure of AriE^D128A. Structure-guided mutagenesis identified W73 as a key regulatory hotspot controlling product diversity, with its variants generating diverse diterpene products. Y203A converted AriE into a highly specific (-)-(R)-cembrene A synthase. Our findings provide the first structural insights into this type of terpene synthase and demonstrate the feasibility of engineering EC variants for selective product formation.