Functional Characterization and Cyclization Mechanism of a Diterpene Synthase Catalyzing the Skeleton Formation of Cephalotane‐Type Diterpenoids

CsCTS, a new diterpene synthase from Cephalotaxus sinensis responsible for forming cephalotene, the core skeleton of cephalotane‐type diterpenoids with a highly rigid 6/6/5/7 tetracyclic ring system, was functionally characterized. The stepwise cyclization mechanism is proposed mainly based on structural investigation of its derailment products, and further demonstrated through isotopic labeling experiments and density functional theory calculations. Homology modeling and molecular dynamics simulation combined with site‐directed mutagenesis revealed the critical amino acid residues for the unique carbocation‐driven cascade cyclization mechanism of CsCTS. Altogether, this study reports the discovery of the diterpene synthase that catalyzes the first committed step of cephalotane‐type diterpenoid biosynthesis and delineates its cyclization mechanism, laying the foundation to decipher and artificially construct the complete biosynthetic pathway of this type diterpenoids.