Epizingiberene synthase: structure, mechanism and a template for design of bioactive chemical space underpinning insect olfaction

萜烯合成酶一起从一个小的非手性底物池产生最大和最多样化的天然产物家族。因此，它们是酶催化研究的典范，可以说是自然界已知的最复杂的单步反应。它们催化一个高度复杂的碳阳离子级联反应，涉及到底物中多达一半碳原子的连通性和杂化变化，通常以惊人的选择性区分能量非常相似的中间物质。直到最近，倍半萜都被认为起源于一个底物（E，E-法尼基二磷酸，EE-FDP），但最近发现了一类新的倍半萜合成酶，它们只使用ZZ-FDP作为底物。我们将使用7-epizingiberene合成酶（EZS）对这类令人兴奋的新酶进行首次机制研究。利用修饰的zz - fdp，位点定向诱变和结构研究将更完整地揭示这一重要酶家族的底物和催化剂之间的复杂相互作用，填补我们对萜烯生物合成知识的明显空白。此外，EZS的产品7-epizingiberene和(R)-curcumene是全球主要农业和园艺作物害虫白蝇的驱避剂。外部化学信号（半化学物质）介导生物体之间的许多相互作用。这些是典型的低分子量亲脂化合物，如倍半萜。一旦释放，这些信号可以通过改变受体生物的行为或发育来起作用。结构类似物将解决与它们的使用相关的许多问题，因为它们可能具有增强的功效和稳定性。然而，直到最近，还没有出现合理的方法来进行SAR研究，因为符号化学物质的受体系统具有极强的选择性，因为它们必须从巨大的多样性和外部化学物质的浓度范围中进行选择（参见可以设计类似物的生物体内的受体系统）。在之前的一项研究中，我们证明了germacrene D类似物的生物活性空间可以通过使用germacrene D合成酶（GDS）来决定。germacrene D是一种由作物在胁迫下释放的倍半萜，具有驱虫作用。这支持了我们的假设，即GDS接受的FDP类似物将导致产品保留活性所需的结构特征。然而，我们开发的这种方法的通用性尚未得到充分测试，因此作为该项目的重要附加价值，我们将使用EZS来生产下一代生物活性白蝇驱蚊剂。这项工作的成功将确保英国合成生物学继续在国际上处于领先地位，并导致对作物保护和粮食安全无害的方法。因此，该项目的总体目标是充分表征一类新的倍半萜合成酶的成员，并具有生产7-epizingiberene和(R)-curcumene的生物活性类似物的额外好处，代表第二代合成半化学品，其化学空间受生物合成的限制。具体目标包括：1)使用已建立的实验室细菌系统生产纯化酶；2)开发新的化学方法，合成可添加到酶制剂中的ZZ-FDP类似物；3)利用未修饰的EZS将合成的底物类似物转化为7-异戊二烯和(R)-姜黄烯类似物，并阐明催化机理；4)进行结构研究和定点诱变以阐明EZS的作用模式，并使用修饰的EZS将合成范围更广的ZZ-FDP底物转化为类似物；5)使用昆虫触角的电记录（电生理学）和实验室行为分析来测量与白蝇物种产生的类似物的活动。

Terpene synthases together generate the largest and most diverse family of natural products from a small pool of achiral substrates. They are hence a paradigm in the study of enzymatic catalysis performing arguably the most complex single-step reactions known in nature. They catalyse a highly intricate carbocationic reaction cascade involving changes in connectivity and hybridisation of up to half the carbon atoms of the substrate, often distinguishing between intermediate species of very similar energy with phenomenal selectivity. Until recently the sesquiterpenes were all thought to originate from just one substrate (E,E-farnesyl diphosphate, EE-FDP) but recently a new class of sesquiterpene synthases that employ ZZ-FDP exclusively as substrate has been discovered. We will perform the first mechanistic investigation of this exciting new class of enzyme using 7-epizingiberene synthase (EZS). Use of modified ZZ-FDPs, site directed mutagenesis and structural studies will reveal more completely the complex interplay between substrate and catalyst for this important family of enzymes, closing a clear gap in our knowledge of terpene biosynthesis.Moreover the products of EZS, 7-epizingiberene and (R)-curcumene, are repellents for whiteflies, major global agricultural and horticultural crop pests. External chemical signals (semiochemicals) mediate many interactions between organisms. These are typically low molecular weight lipophilic compounds such as sesquiterpenes. Upon release, such signals can act by modifying either the behaviour or the development of recipient organisms. Structural analogues would solve many of the problems associated with their use since they may have enhanced efficacy and stability. However, until recently no rational approach has emerged by which to conduct SAR studies since receptor systems for semiochemicals are extremely selective, since they must select from an enormous diversity and concentration range of external chemicals, (cf. receptor systems within organisms for which analogues can be designed). In a previous study, we demonstrated that the bioactive space of analogues of germacrene D, a sesquiterpene that is released by crop plants under stress and which repels aphid populations, could be dictated by use of germacrene D synthase (GDS). This supported our hypothesis that FDP analogues accepted by GDS would lead to products retaining the required structural features for activity. The generality of this approach developed by us has yet to be fully tested however and so as important added value to this project we will use EZS to generate a further generation of bioactive whitefly repellents.Success in this work will ensure the continued leading international presence of UK synthetic biology and lead to environmentally benign approaches to crop protection and food security.Thus, the overall aims of the project are to fully characterise a member of a new class of sesquiterpene synthases with the added benefit of producing biologically active analogues of 7-epizingiberene and (R)-curcumene, representing a second generation of synthetic semiochemicals whose chemical space is dictated by the constraints of biosynthesis. The specific objectives include: 1) production of the purified enzymes using an established laboratory bacterial system; 2) develop novel chemistry to produce synthetic ZZ-FDP analogues that can be added to enzyme preparations; 3) convert synthetic substrate analogues to 7-epizingiberene and (R)-curcumene analogues using unmodified EZS and elucidate the catalytic mechanism; 4) Perform structural studies and site-directed mutagenesis for elucidation of the mode of action of EZS plus use modified EZSs to convert synthetic an extended range of ZZ-FDP substrates to analogues; 5) use electrical recordings of the antennae of insects (electrophysiology), and laboratory behavioural assays, to measure the activity of the generated analogues with whitefly species.