Mechanisms of Insect Pheromone-Binding Proteins and a Bacterial Cytochrome P450: Elucidation of Function and New Applications

昆虫信息素结合蛋白和细菌细胞色素 P450 的机制：功能阐明和新应用

• 批准号: RGPIN-2015-06088
• 负责人: Plettner, Erika
• 金额: $ 4.3万
• 依托单位: Simon Fraser University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=662313
• 关键词: Mechanisms; Insect; Pheromone; Binding; Proteins

My research program targets aspects of insect control that must be addressed to replace insecticides that are not effective/pollute the environment; namely, the development of compounds that interfere with vital olfaction-guided behaviours of insects and the removal of recalcitrant organochlorine insecticides from the environment. Toward this long-term goal, this proposal covers two themes: 1) the role of pheromone-binding proteins (PBPs) in pheromone olfaction in moths, and 2) new reactions catalyzed by cytochrome P450cam. ***Insects use species-specific signal chemicals (pheromones) to communicate with each other; e.g. gypsy moth females emit a chiral epoxide pheromone that attracts males. PBPs are the first insect-specific protein in the olfactory hairs of insect antennae to interact selectively with odorants. PBPs are essential to olfaction, yet how they contribute to this sense is poorly understood. We propose to determine the structure of two gypsy moth PBPs, and study the interaction of these two proteins with the pheromone, analogs and endogenous substances from olfactory hairs. Results of thermodynamic and kinetic studies will be correlated with electrophysiological data to link molecular interactions of PBPs and their ligands with quantifiable olfactory responses that trigger vital behaviours.***Cytochrome P450cam is an enzyme that catalyzes the oxidation of the monoterpene camphor, using oxygen from air. This is the first step of a camphor degradation pathway used by a strain of Pseudomonas putida, a common soil bacterium. My group has found that, under anoxic conditions, this enzyme catalyzes the reduction of camphor to borneol, and that borneol formed regulates the expression of P450cam and its two partner proteins. We will study this unusual reduction in more detail. Additionally, we will examine mutants of P450cam that remove chlorine atoms from 3-chloroindole or from endosulfan diol. The former substrate forms a pigment upon oxidation and removal of chloride, which enables us to obtain kinetic data. Some of these mutants will be studied further as potential biocatalysts for the formation of pyrroloindoles, bitter compounds that could be used , e.g., as gypsy moth larval feeding deterrents. Endosulfan diol is derived from the hexachlorinated insecticide endosulfan (ES), which has been used worldwide since the 1950s. ES and the diol have accumulated in the environment, because their hexachlorinated carbon framework is difficult for enzymes to destroy. Though ES is being phased out by 2016, the legacy of its use to control insects will remain for some time. Hence, we propose to study mutants (discovered by my group) that dechlorinate ES diol.***An understanding of these systems is highly useful in the development of new insect control agents or biocatalysts that could be applied to the biodegradation of persistent polychlorinated pollutants or the synthesis of new compounds.**

我的研究计划针对昆虫控制的方面，必须解决取代杀虫剂是无效/污染环境;即，干扰昆虫的重要嗅觉引导行为的化合物的发展和从环境中去除有害的有机氯杀虫剂。为了实现这一长期目标，本研究计划包括两个主题：1）信息素结合蛋白（PBP）在蛾类信息素嗅觉中的作用，以及2）细胞色素P450 cam催化的新反应。 * 昆虫使用物种特异性信号化学物质（信息素）相互交流;例如，舞毒蛾雌性释放吸引雄性的手性环氧信息素。PBPs是昆虫触角嗅毛中第一个与气味物质选择性相互作用的昆虫特异性蛋白。 PBPs对于嗅觉是必不可少的，但它们如何有助于这种感觉却知之甚少。 我们拟确定舞毒蛾两种PBP的结构，并研究这两种蛋白与信息素、类似物和嗅毛内源物质的相互作用。 热力学和动力学研究的结果将与电生理数据相关联，以将PBPs及其配体的分子相互作用与触发生命行为的可量化嗅觉反应联系起来。细胞色素P450 cam是一种酶，它利用空气中的氧气催化单萜樟脑的氧化。 这是恶臭假单胞菌（一种常见的土壤细菌）降解樟脑途径的第一步。 我的研究小组发现，在缺氧条件下，这种酶催化樟脑还原为樟脑，而樟脑的形成调节P450 cam及其两个伴侣蛋白的表达。我们将更详细地研究这种不寻常的减少。 此外，我们将研究P450 cam的突变体，从3-氯吲哚或硫丹二醇中去除氯原子。 前一种基质在氧化和除去氯化物后形成颜料，这使我们能够获得动力学数据。 这些突变体中的一些将被进一步研究，作为形成吡咯并吲哚的潜在生物催化剂，可以使用的苦味化合物，例如，作为舞毒蛾幼虫的摄食抑制剂。硫丹二醇源自六氯杀虫剂硫丹，自20世纪50年代以来在全世界使用。 ES和二醇在环境中积累，因为它们的六氯碳框架很难被酶破坏。 虽然ES将在2016年被淘汰，但其用于控制昆虫的传统仍将持续一段时间。 因此，我们建议研究使ES二醇脱氯的突变体（由我的小组发现）。对这些系统的了解对于开发可用于持久性多氯污染物生物降解或合成新化合物的新的昆虫控制剂或生物催化剂非常有用。