Insect cuticular lipid degradation: characterization of cytochrome P450 monooxygenases from the entomopathogenic fungus Beauveria bassiana

HUARTE BONNET CARLA; KEYHANI NEMAT O; JUÁREZ M. PATRICIA; PEDRINI N.

Congreso; 45th Annual Meeting of the Society for Invertebrate Pathology.; 2012

Cytochrome P450 monooxygenases constitute a superfamily of heme-thiolate proteins that act on a wide variety of endogenous and xenobiotic molecules. Very long chain hydrocarbons, a major component of the outer insect waxy layer, represent the substrates for a specific subset of these enzymes that participate in hydrocarbon degradation. At least eight cytochrome P450s (CYP) genes potentially involved in lipid metabolism were previously characterized in the entomopathogenic fungus Beauveria bassiana. Hydrocarbons and insect cuticular lipids induced the expression of these genes. One B. bassiana cytochrome P450 (CYP52X1) was able to oxidize long chain fatty acids. In this work, four additional B. bassiana cytochrome P450s: CYP5337A1, CYP52G11, CYP53A26, and CYP584Q, containing C-terminal poly-His tag fusions were expressed in the yeast Saccharomyces cerevisiae strain WAT11. After induction, the yeast cells were harvested and endoplasmic reticulum-derived microsomes were obtained. Total membrane proteins were solubilized by treatment with sodium cholate, and loaded onto columns containing nickel-charged resin for purification of the His-tagged proteins. The molecular masses of the purified proteins were estimated by SDS-PAGE and total cytochrome P450 content was measured by spectrophotometry. These results, together with ongoing experiments on substrate specificity, will help characterize the biochemical properties of the cytochrome P450s of B. bassiana leading to better understanding their roles in insect lipid degradation.