Xing Baiying, Yu Jiahui, Chi Changbiao, Ma Xueyang, Xu Qingxia, Li Annan, Ge Yuanjie, Wang Zhengdong, Liu Tan, Jia Hongli, Yin Fuling, Guo Juan, Huang Luqi, Yang Donghui, Ma Ming
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Haidian District, Beijing, 100191, China.
State Key Laboratory of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
Commun Chem. 2021 Sep 30;4(1):140. doi: 10.1038/s42004-021-00578-z.
Pimarane-type diterpenoids are widely distributed in all domains of life, but no structures or catalytic mechanisms of pimarane-type diterpene synthases (DTSs) have been characterized. Here, we report that two class I DTSs, Sat1646 and Stt4548, each accept copalyl diphosphate (CPP) as the substrate to produce isopimara-8,15-diene (1). Sat1646 can also accept syn-CPP and produce syn-isopimaradiene/pimaradiene analogues (2-7), among which 2 possesses a previously unreported "6/6/7" ring skeleton. We solve the crystal structures of Sat1646, Sat1646 complexed with magnesium ions, and Stt4548, thereby revealing the active sites of these pimarane-type DTSs. Substrate modeling and subsequent site-directed mutagenesis experiments demonstrate different structural bases of Sat1646 and Stt4548 for 1 production. Comparisons with previously reported DTSs reveal their distinct carbocation intermediate stabilization mechanisms, which control the conversion of a single substrate CPP into structurally diverse diterpene products. These results illustrate the structural bases for enzymatic catalyses of pimarane-type DTSs, potentially facilitating future DTS engineering and combinatorial biosynthesis.
半日花烷型二萜类化合物广泛分布于生物的各个领域，但半日花烷型二萜合酶（DTSs）的结构或催化机制尚未得到表征。在此，我们报道了两个I类DTS，Sat1646和Stt4548，它们均以柯巴基二磷酸（CPP）为底物生成异海松-8,15-二烯（1）。Sat1646还能接受顺式-CPP并生成顺式异海松二烯/海松二烯类似物（2-7），其中2具有先前未报道的“6/6/7”环骨架。我们解析了Sat1646、与镁离子复合的Sat1646以及Stt4548的晶体结构，从而揭示了这些半日花烷型DTS的活性位点。底物建模及随后的定点诱变实验证明了Sat1646和Stt4548生成1的不同结构基础。与先前报道的DTSs比较揭示了它们不同的碳正离子中间体稳定机制，这些机制控制了单一底物CPP向结构多样的二萜产物的转化。这些结果阐明了半日花烷型DTSs酶促催化的结构基础，可能有助于未来的DTS工程和组合生物合成。
