Abstract：
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 (Mg2+), 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.
摘要：
对塞萜合酶（StTSs）的结构和催化机理知之甚少，这极大地阻碍了StTSs的基于结构的工程，以扩展酯的结构多样性。我们在这里报告了两个真菌StTS的萜烯环化（TC）结构域的晶体结构：酯渔醇合酶（NfSS）和酯碳三烯合酶（PbSS）。两种TC结构都含有苄基三乙基氯化铵（BTAC），焦磷酸盐(PPi)，和镁离子（Mg2+），明确定义催化活性位点。理论和实验的结合，包括碳阳离子中间体建模，定点诱变，同位素标记为其催化机理的结构基础提供了详细的见解。NfSS和PbSS的基于结构的工程导致20种酯烯的形成，包括13种新化合物和4对在C18具有不同构型的差向异构体。这些结果扩展了酯烯的结构多样性，并为未来的合成生物学研究提供了重要见解。
