Discovery of Class I Diterpene Cyclases Producing a Tetracyclic Cephalotene Skeleton in Plum Yews
Yaping Mao, Guangyi Wang, Jianhua Li, Zhanguang Feng, Yuhong Ren, Yu-Wei Sun, Yong Wang
Abstract
The cephalotane diterpenoids (e.g., harringtonolide, cephanolides) from plum yews ( Cephalotaxus ) represent a rare class of tetracyclic C 20 and C 19 natural products with intriguing bioactivities. Here, we report the discovery and functional characterization of multiple diterpene synthases (diTPSs) from Cephalotaxus harringtonia and Cephalotaxus hainanensis . In addition to diTPSs responsible for ent -kaurene and biformene biosynthesis, two class I cephalotene synthases (CSs) were found to directly cyclize geranylgeranyl diphosphate (GGPP) into a unique cephalot-12-ene skeleton, which is a crucial step in the biogenesis of cephalotane diterpenoids. A plausible biosynthetic mechanism en route to tetracyclic ring formation was proposed based on an isotope labeling experiment combined with quantum chemical calculations. Site-directed mutagenesis indicated that residue F625 in CS is a key switch of diversifying the hydrocarbon backbone to bicyclic verticillene and tricyclic taxadiene. Altogether this report provides insight into the carbocation cascade reaction driven by active sites in class I diTPSs, promotes our understanding of how chemical diversity originates and evolves in gymnosperms, and lays a solid foundation for future investigation of Cephalotaxus diterpene metabolites.