Gene Engineering and Biotechnology Beijing Key Laboratory, College of Life Sciences, Beijing Normal University, Beijing, 100875, China.
Gene Engineering and Biotechnology Beijing Key Laboratory, College of Life Sciences, Beijing Normal University, Beijing, 100875, China.
Phytochemistry. 2021 Jan;181:112573. doi: 10.1016/j.phytochem.2020.112573. Epub 2020 Nov 1.
Terpenes form a class of highly diverse natural products. The diversity of terpenes is created by terpene synthases. During the reaction, carbocation intermediates form and their rearrangement could lead to the formation of various products. Terpene synthases determine the final product profile by controlling the conformation of the intermediate or stabilizing the carbocation. Pinene synthase is a monoterpene synthase catalyzing the cyclization of geranyl pyrophosphate (GPP) to form pinene. Our study suggests that by mutating the aromatic residue F482 to Ala, Val, Ile and Thr, the enzyme can be converted to sabinene synthase, with more than 90% of its total terpene products becoming sabinene, which indicates the aromaticity of this residue is essential for stabilizing the pinyl carbocation. We also identified a mutation S491A that could cause an about 29% increase in the overall activity of the enzyme without altering its produce selectivity. Molecular dynamic simulation indicates this mutation could decrease the flexibility of the enzyme when it forms a complex with the pinyl carbocation. Our study suggests the active pocket of pinene synthase has a certain level of plasticity, making it relatively easy to change the product selectivity or overall activity. This property could have an important implication in the evolution of terpene synthases and thereby terpene diversity, as by changing a few residues an enzyme could synthesize a completely different terpene product in a significant amount, which allows selection to take place.
萜类化合物形成了一类高度多样化的天然产物。萜类化合物的多样性是由萜烯合酶创造的。在反应过程中，形成碳正离子中间体，其重排可能导致各种产物的形成。萜烯合酶通过控制中间体的构象或稳定碳正离子来决定最终的产物分布。β-蒎烯合酶是一种单萜合酶，催化香叶基焦磷酸（GPP）的环化形成β-蒎烯。我们的研究表明，通过将芳香残基 F482 突变为丙氨酸、缬氨酸、异亮氨酸和苏氨酸，该酶可以转化为柠檬烯合酶，其超过 90%的总萜烯产物成为柠檬烯，这表明该残基的芳香性对于稳定烯丙基碳正离子是必不可少的。我们还鉴定了一个突变 S491A，它可以在不改变产物选择性的情况下使酶的总活性增加约 29%。分子动力学模拟表明，该突变可能会降低酶与烯丙基碳正离子形成复合物时的灵活性。我们的研究表明，β-蒎烯合酶的活性口袋具有一定的可塑性，使得改变产物选择性或总活性相对容易。这种特性可能对萜烯合酶的进化以及萜烯的多样性具有重要意义，因为通过改变几个残基，一种酶可以以大量的方式合成完全不同的萜烯产物，从而允许选择发生。
