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

Epiizingiberene合酶：结构、机制和用于设计支撑昆虫嗅觉的生物活性化学空间的模板

基本信息

批准号：
BB/M022463/1
负责人：
Rudolf Allemann
金额：
$ 58.69万
依托单位：
CARDIFF UNIVERSITY
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2015
资助国家：
英国
起止时间：
2015 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FM022463%2F1
关键词：
Epizingiberene synthase structure mechanism template

项目摘要

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.

萜烯脱氢酶共同从少量非手性底物中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，可以说是自然界中已知的最复杂的一步反应。它们催化一个高度复杂的碳阳离子反应级联，涉及底物中多达一半碳原子的连接性和杂化的变化，通常以惊人的选择性区分能量非常相似的中间物种。直到最近，倍半萜烯都被认为仅来源于一种底物（E，E-法呢基二磷酸，EE-FDP），但最近发现了一类专门使用ZZ-FDP作为底物的新的倍半萜烯脱氢酶。我们将使用7-表姜烯合酶（EZS）对这种令人兴奋的新酶进行首次机理研究。使用修饰的ZZ-FDP，定点突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，填补了我们在萜烯生物合成方面的知识空白。此外，EZS的产物7-表姜烯和（R）-姜黄烯是全球主要农业和园艺作物害虫粉虱的驱避剂。外部化学信号（化学信息素）介导生物体之间的许多相互作用。这些通常是低分子量亲脂性化合物，例如倍半萜烯。一旦释放，这些信号可以通过改变受体生物体的行为或发育来发挥作用。结构类似物将解决与其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，还没有出现进行SAR研究的合理方法，因为化学信息素的受体系统具有极强的选择性，因为它们必须从外部化学物质的巨大多样性和浓度范围中进行选择。生物体内的受体系统，可以为其设计类似物）。在以前的研究中，我们证明了大根香叶烯D的类似物的生物活性空间，一种倍半萜烯，在压力下由作物植物释放，并排斥蚜虫种群，可以通过使用大根香叶烯D合酶（GDS）来决定。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。我们开发的这种方法的通用性还没有得到充分的测试，但是，作为该项目的重要附加值，我们将使用EZS产生下一代生物活性粉虱驱避剂。这项工作的成功将确保英国合成生物学继续保持领先的国际地位，并导致对作物保护和粮食安全的环境友好的方法。因此，该项目的总体目标是充分利用一类新的倍半萜烯脱氢酶的成员，其附加益处是产生7-表姜烯和（R）-姜黄烯的生物活性类似物，代表第二代合成化学信息素，其化学空间由生物合成的限制决定。具体目标包括：1）使用已建立的实验室细菌系统生产纯化的酶; 2）开发新的化学方法以生产可以添加到酶制剂中的合成的ZZ-FDP类似物; 3）使用未修饰的EZS将合成的底物类似物转化为7-表姜烯和（R）-姜黄烯类似物并阐明催化机制; 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将合成的扩展范围的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）和实验室行为测定来测量所产生的类似物与粉虱物种的活性。