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, four eunicellane cyclases (ECs) have been reported: cis-ECs (Bnd4 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 into 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. L96V specifically produced 1, while 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.
Supplementary materials
Title
Structural Insights into the Catalytic Mechanism of cis-Eunicellane Cyclase AriE
Description
Materials and methods, detailed experimental procedures, and one- and two-dimensional NMR data of all isolated compounds.
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