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

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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.**