State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Haidian District, Beijing, 100191, China.
Kekulé Institute of Organic Chemistry and Biochemistry, University of Bonn, Gerhard-Domagk-Strasse 1, 53121, Bonn, Germany.
Angew Chem Int Ed Engl. 2024 Jun 3;63(23):e202405140. doi: 10.1002/anie.202405140. Epub 2024 May 2.
Little is known about the structures and catalytic mechanisms of sesterterpene synthases (StTSs), which greatly hinders the structure-based engineering of StTSs for structural diversity expansion of sesterterpenes. We here report on the crystal structures of the terpene cyclization (TC) domains of two fungal StTSs: sesterfisherol synthase (NfSS) and sesterbrasiliatriene synthase (PbSS). Both TC structures contain benzyltriethylammonium chloride (BTAC), pyrophosphate (PPi), and magnesium ions (Mg), clearly defining the catalytic active sites. A combination of theory and experiments including carbocationic intermediates modeling, site-directed mutagenesis, and isotope labeling provided detailed insights into the structural basis for their catalytic mechanisms. Structure-based engineering of NfSS and PbSS resulted in the formation of 20 sesterterpenes including 13 new compounds and four pairs of epimers with different configurations at C18. These results expand the structural diversity of sesterterpenes and provide important insights for future synthetic biology research.
关于甾体萜烯合酶（StTSs）的结构和催化机制知之甚少，这极大地阻碍了基于结构的 StTSs 工程改造，以扩大甾体萜烯的结构多样性。我们在此报告了两种真菌 StTSs 的萜烯环化（TC）结构域的晶体结构：海松醇合酶（NfSS）和巴西三烯合酶（PbSS）。这两个 TC 结构都包含苄基三乙基氯化铵（BTAC）、焦磷酸（PPi）和镁离子（Mg），明确了催化活性位点。理论和实验的结合，包括碳正离子中间体建模、定点突变和同位素标记，提供了对其催化机制结构基础的详细了解。基于结构的 NfSS 和 PbSS 工程改造导致了 20 种甾体萜烯的形成，包括 13 种新化合物和 4 对 C18 构型不同的差向异构体。这些结果扩展了甾体萜烯的结构多样性，并为未来的合成生物学研究提供了重要的见解。
