Unexpected Main-Chain-Mediated and Neutral-Intermediate-Involved Catalytic Reaction in a High-Fidelity ent-Neoabietadiene Synthase

Labdane-related diterpenoids (LRDs) represent a large group of terpenoids. Their diverse skeletons arise from the complex cyclization reactions, which are catalyzed by LRD diterpene synthases (diTPSs) using geranylgeranyl pyrophosphate (GGPP) as the common precursor and involve a multistep cascade of cyclization and rearrangement. So far, the catalytic mechanisms of LRD diTPSs still remain largely unknown. Herein, we characterized five LRD diTPSs from Euphorbia fischeriana Steud. Notably, EfTPS14 is a rare high-fidelity ent-neoabietadiene synthase, playing a vital role in the biosynthesis of jolkinolides, a group of anticancer LRDs. Its catalytical pathway involves a neutral intermediate ent-sandaracopimaradiene and a deprotonation-reprotonation sequence, which is directly mediated by the carbonyl oxygen of I644 located in the G1/2 break region. Moreover, we found that L765 and L762 are responsible for regioselective deprotonation to generate ent-neoabietadiene. Such a main-chain-mediated and neutral intermediate-involved pathway enriches our understanding of the catalytic mechanism of diTPSs and lays a foundation for altering their catalytic properties.