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

萜烯合成酶共同从一小群非手性底物中产生最大和最多样化的天然产物家族。因此，它们是酶催化研究中的一个范例，可以说是自然界中最复杂的单步反应。它们催化一个高度复杂的碳阳离子反应级联反应，涉及底物多达一半碳原子的连接性和杂化作用的变化，经常区分能量非常相似的中间物种，具有惊人的选择性。直到最近，倍半萜都被认为只起源于一种底物(E，E-法尼基二磷酸，EE-FDP)，但最近发现了一类新的以ZZ-FDP为底物的倍半萜合成酶。我们将使用7-表木烯合成酶(EZs)对这类令人兴奋的新酶进行第一次机制研究。使用修饰的ZZ-FDPs、定点突变和结构研究将更全面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，填补我们在萜类生物合成方面的知识空白。此外，EZ的产物7-表麻黄烯和(R)-姜黄烯是全球主要农业和园艺作物害虫烟粉虱的驱避剂。外部化学信号(信号化学物质)在生物体之间的许多相互作用中起中介作用。这些是典型的低分子亲油化合物，如倍半萜。一旦释放，这种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有关的许多问题，因为它们可能具有更高的效力和稳定性。然而，直到最近，还没有出现一种合理的方法来进行SAR研究，因为信号化学物质的受体系统具有极高的选择性，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(参看。生物体内可设计类似物的受体系统)。在以前的研究中，我们证明了杰马卡烯D的类似物的生物活性空间可以通过使用杰马卡林D合成酶(GDS)来决定。杰马卡林D是一种由作物在胁迫下释放的倍半萜类化合物，它可以驱避蚜虫种群。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普适性还没有得到充分的测试，所以作为这个项目的重要附加值，我们将使用EZ来产生新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在国际上处于领先地位，并导致对环境友好的作物保护和食品安全方法。因此，该项目的总体目标是充分描述一类新的倍半萜合成酶的成员的特征，以及生产具有生物活性的类似物7-表辛赤烯和(R)-姜黄烯的额外好处，这是第二代合成半导体化合物，其化学空间由生物合成的限制决定。具体目标包括：1)利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产生可添加到酶制剂中的合成ZZ-FDP类似物；3)使用未经修饰的EZ将合成底物类似物转化为7-表麻黄烯和(R)-姜黄烯类似物，并阐明其催化机理；4)进行结构研究和定点突变以阐明EZ的作用方式，并使用经修饰的EZSS将合成的更广泛的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，以测量产生的类似物与粉虱物种的活动。

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.