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

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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.