Mechanistic Characterization of Diterpene Synthase Pairs for Tricyclic Diterpenes from Cyanobacteria

作者：Jiayi Yu, Taro Shiraishi, Kizerbo A. Taizoumbe, Yusaku Karasuno, Ayako Yoshida, Makoto Nishiyama, Jeroen S. Dickschat, Tomohisa Kuzuyama · 发表于：Journal of the American Chemical Society · 年份：2025 · DOI：10.1021/jacs.4c16710 · 被引用次数：9 · 研究领域：Plant biochemistry and biosynthesis、Antioxidant Activity and Oxidative Stress、Microbial Natural Products and Biosynthesis

In recent years, genome mining in cyanobacteria has revealed abundant gene clusters related to natural product biosynthesis. However, only a few terpene synthases (TSs) have been identified from this bacterial phylum. Pfam profiles, such as PF03936 and PF19086, which are frequently used for TS retrieval, are built from plant, bacterial, and fungal TSs. Herein, we constructed a new hidden Markov model (HMM) specific to bacterial TSs on the basis of 110 bacterial TSs experimentally validated in recent years. Using this model, we identified a pair of diterpene synthases, Cpt11 (class II TS) and Cts11 (class I TS), in the cyanobacterium Scytonema tolypothrichoides . In vitro experiments demonstrated that Cpt11 catalyzes the formation of syn -copalyl diphosphate from geranylgeranyl diphosphate and that Cts11 subsequently converts syn -copalyl diphosphate into a rare 6,6,7-tricyclic diterpene alcohol. Its biosynthesis was established through isotope labeling experiments, which revealed a unique sequence of a 1,6-proton shift and ring expansion to a seven-membered ring. We solved the crystal structure of Cts11 at a resolution of 1.76 Å. Additionally, via site-directed mutagenesis experiments, we identified two amino acid residues whose exchanges affected the formation of the original diterpene alcohol, leading to the formation of two new compounds: a 6,6,7-tricyclic diterpene hydrocarbon and another 6,6,6-tricyclic diterpene alcohol. A BLAST search revealed several sequences that sh...