Structure and Mechanisms of Styrene Monooxygenase

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): Styrene is an important component of a wide array of plastic, rubber, and over the counter adhesive products. Over the past century, large-scale industrial and shipping accidents and inappropriate waste disposal practices have caused aquatic and terrestrial environments to become heavily contaminated with monomeric styrene. In the work place, individuals involved in both the formulation and application of styrene-based materials are at the greatest risk of high levels exposure. As a biochemical toxin, styrene induces the activity of iron and flavin-dependent monooxygenases, which catalyze epoxidation and hydroxylation reactions to yield strong alkylating agents and pulmonary irritants such as styrene oxide and vinyl phenols. The long-term objective of our research is to elucidate the structures and mechanisms of the enzymes engaged in the styrene metabolic pathway and to establish a model that allows a more accurate evaluation of the human health risk associated with exposure. Our work will also provide a framework for studies of other metabolic and detoxification pathways, which include the synthesis of toxic or unstable pathway intermediates. The enzymes of the styrene metabolic pathway, styrene monooxygenase, styrene oxide isomerase, and phenacetaldehyde dehydrogenase, have been cloned and will be investigated through mechanistic and structural studies. Styrene monooxygenase will be functionally characterized in our laboratory and structurally characterized through X-ray crystallography by Dr. Amy Rosenzweig's group at Northwestern University. Single-turnover and steady-state kinetic studies will be used to characterize the intermediates involved in the styrene epoxidation reaction and to establish the role of protein-protein interactions in the modulation of reaction rates. A combination of stopped-flow and rapid quench studies will be conducted to establish the mechanisms and efficiency of reactive substrate and coenzyme transport in styrene metabolism. Diffraction quality crystals of styrene monooxygenase will be solved and used to identify active site structures engaged catalysis. This work will result in the first complete structural and mechanistic evaluation of a flavoprotein epoxidase. Elucidation of the enzyme structures and mechanisms engaged in the generation and shuttling of the toxic intermediates during styrene metabolism is an essential step in identifying the health risks associated with human and environmental exposure to monomeric styrene.