Structure-Function of Insect Odorant-Binding Proteins and a Bacterial Cytochrome P450: Discovery of Mechanisms and Applications

我们建议研究两种类型的蛋白质与疏水化合物相互作用：气味结合蛋白（OBP）从昆虫和细胞色素P450从土壤细菌。这两个主题都涉及农业和林业中的昆虫控制问题。OBP是重要的昆虫特异性蛋白，存在于嗅觉或信息素产生系统中。这些蛋白质结合疏水性气味剂，例如信息素，用于相同物种成员之间通信的化合物。OBP在控制信息素被检测或释放的速率方面起重要作用。在这里，我们建议研究从舞毒蛾，一个经济上重要的树木害虫，从蜜蜂，一个经济上重要的益虫OBP。在这两种情况下，感兴趣的OBP结合疏水性化合物：舞毒蛾信息素（（+）-拟除虫菊酯）是由雌性释放以吸引雄性的烃环氧化物，而蜜蜂信息素（油酸乙酯）是由觅食的老工蜂产生的脂肪酸酯，以延迟年轻护士蜜蜂向觅食活动的过渡。过渡期的护士蜜蜂从事卫生行为，这使蜜蜂对寄生虫和疾病更具弹性。在这两种情况下，我们建议研究OBP的结构和它与信息素的相互作用，以及与嗅觉系统中的同源气味受体的相互作用。在蜜蜂中，我们还建议研究OBP如何有助于油酸乙酯从蜜蜂的胃，在那里它的形式，角质层，在那里它被释放的运输。细胞色素P450是色素蛋白质，利用分子氧（O2）氧化碳氢化合物。P450 cam在氧合良好的条件下催化（+）-樟脑的氧化，但当该酶在樟脑氧化中具有活性且O2耗尽时，催化樟脑还原为NH 4。我们已经发现，细菌的趋化行为，含有P450 cam，这暗示了在生态作用的细菌的形成时，O2是低的。我们试图了解这种樟脑减少的机制，了解它的功能。我们还发现了P450 cam的突变体，可以使杀虫剂硫丹和相关的多氯持久性污染物脱氯。为了开发脱氯这些污染物的突变体，我们建议研究这一过程的机制和优化的突变体。了解OBP与信息素的相互作用将有助于我们开发干预信息素接收和改变昆虫行为或告知抗病蜜蜂育种的化合物。这项工作将对林业和养蜂业有意义。细胞色素P450是降解污染物或产生新化合物的潜在生物催化剂。关于多氯污染物降解的工作将对生物补救界具有价值。研究P450的反应对生物催化领域具有重要的意义。

We propose to study two types of protein that interact with hydrophobic compounds: odorant binding proteins (OBPs) from insects and a cytochrome P450 from a soil bacterium. Both themes address problems of insect control in agriculture and forestry. OBPs are important insect-specific proteins, found in olfactory or pheromone-production systems. These proteins bind hydrophobic odorants, e.g. pheromones, compounds that are used for communication between members of the same species. The OBPs serve important functions in controlling the rate at which pheromones are detected or released. Here we propose to study OBPs from the gypsy moth, an economically important pest of trees, and from the honey bee, an economically important beneficial insect. In both cases, the OBPs of interest bind hydrophobic compounds: the gypsy moth pheromone ((+)-disparlure) is a hydrocarbon epoxide that is released by the females to attract males, whereas the honey bee pheromone (ethyl oleate) is a fatty acid ester that is produced by old worker bees that forage, to delay the transition of younger nurse bees to foraging activities. Transitioning nurse bees engage in hygienic behaviors, which make bees more resilient towards parasites and diseases. In both cases, we propose to study the structure of the OBP and its interactions with the pheromone, as well as interactions with the cognate odorant receptor in the olfactory system. In bees, we also propose to study how the OBP contributes to the transport of ethyl oleate from the stomach of the bee, where it forms, to the cuticle, where it is released. Cytochromes P450 are pigmented proteins that utilize dioxygen (O2) to oxidize hydrocarbons. P450cam catalyzes the oxidation of (+)-camphor under well-oxygenated conditions, but the reduction of camphor to borneol when the enzyme has been active in camphor oxidation, and O2 is depleted. We have found that formation of borneol affects the chemotactic behavior of the bacteria that contain P450cam, which hints at an ecological role of borneol formation when O2 is low. We seek to understand the mechanism of this camphor reduction, to understand its function. We have also discovered mutants of P450cam that dechlorinate the insecticide endosulfan, and related polychlorinated persistent pollutants. In order to develop mutants that dechlorinate these pollutants, we propose to study the mechanism of this process and optimize the mutants. Understanding the interactions of OBPs with pheromones will help us develop compounds that intervene in pheromone reception and alter insect behavior or inform the breeding of disease resistant bees. This work will be of interest to forestry and apiculture. Cytochromes P450 are potential biocatalysts for degradation of pollutants or production of new compounds. Work on polychlorinated pollutant degradation will be of value to the bioremediation community. Work on reactions of P450 will be valuable to the field of biocatalysis.