Crystal Structure and Catalytic Mechanism of the cis‐Eunicellane Cyclase AriE

Abstract: 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 AriED128A. 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.