Probing sesquiterpene synthase chemistry with non-canonical amino acids

萜类化合物已知超过55000种，在各种生命形式中发挥着无数的功能，是地球上发现的最大、结构和立体化学最多样化的天然产物家族。萜类化合物存在于海洋和陆地植物、真菌、细菌和昆虫中，在农业中有许多应用，如抗真菌剂、除草剂或杀虫剂、化妆品中的香料、抗生素、抗癌剂、激素或药物中的避孕药。尽管结构多样，但所有的萜类化合物都是由两种化学上相关的分子，即delta1-和delta2-异戊烯基二磷酸，生物合成的。这一发现在100多年前由德国化学家奥托·瓦拉赫（Otto Wallach， 1910年诺贝尔奖）首次提出，并由苏黎世的利奥波德·鲁兹卡（Leopold Ruzicka， 1939年诺贝尔奖）奠定了正确的生化基础。二磷酸异戊烯基通过延伸反应连接形成所有萜类化合物的线性前体，根据它们所含的异戊烯基单位（C5）的数量分为单萜（C10）、倍半萜（C15）、二萜（C20）和更高分子量的萜类化合物。萜烯合成酶的作用将这些线性前体转化为大量的环状产物。正是这些环化在很大程度上导致了萜类天然产物的多样性。值得注意的是，所有这些酶都有一个共同的三维折叠，因此最终结构的复杂性和多样性与这些产物的简单性形成了对比。为了破译决定萜烯合成酶特异性的密码，需要对单个反应机制有更深入的了解。我们以前使用的技术是对底物进行修饰或将萜烯合成酶活性位点内的单个氨基酸替换为其他氨基酸，并研究对反应的影响。后一种方法受到这样一个事实的限制，即在自然发生的蛋白质中通常只观察到20种不同的氨基酸。我们现在希望使用一种新技术，允许将非天然氨基酸结合到酶中，可以选择这样一种方式，以获得有关反应机制的特定问题的答案，从而进一步了解这组令人着迷的酶。这项工作最终将为我们提供知识，以产生新的类萜烯，但在基础科学和工业中具有许多潜在应用的非天然分子。

With more than 55,000 known compounds serving myriad functions in all forms of life, the terpenoids are the largest as well as the most structurally and stereochemically diverse family of natural products found on earth. Terpenoids are found in marine and terrestrial plants, fungi, bacteria and insects and have many applications in agriculture as antifungals, herbicides or insecticides, as fragrances in cosmetics, or as antibiotics, anti cancer agents, hormones, or contraceptive agents in medicine. Despite their structural diversity, all terpenoids are biosynthesised from two chemically related molecules, namely delta1- and delta2-isopentenyldiphosphate, an observation which was first made by the German chemist Otto Wallach more than 100 years ago (Nobel prize 1910) and put on the correct biochemical foundation by Leopold Ruzicka in Zurich (Nobel prize 1939). The isopentenyldiphosphates are joined in elongation reactions to form the linear precursors of all terpenoids, which are classified according to the number of isopentenyl-units (C5) they contain as monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20) and higher molecular weight terpenoids. The action of terpene synthases then converts these linear precursors into a large number of cyclic products. It is these cyclisations that are responsible to a large part for the diversity seen in terpenoid natural products. Remarkably all these enzymes share a common three-dimensional fold and hence the complexity and diversity of the final structures is contrasted with the simplicity of the generation of these products. In order to decipher the code that determines the specificity of terpene synthases a much deeper understanding of individual reaction mechanisms is required. We have previously used techniques where the substrates were modified or where individual amino acids within the active site of terpene synthases were replaced with others and the effects on the reaction studied. This latter approach is limited by the fact that only 20 different amino acids are normally observed in naturally occurring proteins. We now wish to use a new technique that allows the incorporation into enzymes of non-natural amino acids that can be chosen in such a way as to obtain answers to specific questions about the reaction mechanisms and hence further our understanding of this fascinating group of enzymes. This work should eventually provide us with the knowledge to generate new terpene-like, but non-natural molecules with many potential applications in basic science and industry.