Discovery of Class I Diterpene Cyclases Producing a Tetracyclic Cephalotene Skeleton in Plum Yews

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.

Structural and Catalytic Insight into the Unique Pentacyclic Triterpene Synthase TwOSC

“…Although multistep endothermic reactions are extremely rare in the formation of terpenoids, a continuous three‐step endothermic reaction has been reported during the cyclization process by a diterpenoid cyclase. It is worth noting that the entire reaction process is still in an exothermic state, which is consistent with our results [19] . In this context, the QM/MM‐mapped relative energy profile of the reaction (Figure 2a) validated the hypothesized complete catalytic process (Figure 1e) from the linear substrate to the final friedelin.…”

“…It is worth noting that the entire reaction process is still in an exothermic state, which is consistent with our results. [19] In this context, the QM/MM-mapped relative energy profile of the reaction (Figure 2a) validated the hypothesized complete catalytic process (Figure 1e) from the linear substrate to the final friedelin. The final product friedelin obtained from the QM/MM scan exhibited an all-chair (c-c-c-c-c) conformation (Fig- 1e).…”

Structural and Catalytic Insight into the Unique Pentacyclic Triterpene Synthase TwOSC

“…It is worth noting that the entire reaction process is still in an exothermic state, which is consistent with our results. [19] In this context, the QM/MM-mapped relative energy profile of the reaction (Figure 2a) validated the hypothesized complete catalytic process (Figure 1e) from the linear substrate to the final friedelin. The final product friedelin obtained from the QM/MM scan an all-chair (c-c-c-c-c) conformation (Figure 1e).…”

Cephalotane diterpenoids: structural diversity, biological activity, biosynthetic proposal, and chemical synthesis

“…Lately, the discovery of diterpene cyclases catalyzing the skeleton formation has addressed the issue. 27,28 In addition, due to the complex cage-like framework and remarkable bioactivities of cephalotane diterpenoids, they are regarded as excellent targets for total synthesis. The past 30 years have witnessed a multitude of synthetic efforts toward this diterpenoid class, 29–49 leading to a battery of elegant completed total syntheses by the groups of Mander, 50 Tang, 51 Zhai, 52–54 Hu, 55–57 Gao, 58–60 Zhao, 61 Cai, 62 Zhang/Yang, 63 and Sarpong.…”