Structure and Mechanisms of Styrene Monooxygenase

描述（由申请人提供）：苯乙烯是各种塑料，橡胶和非处方粘合剂产品的重要组成部分。在过去的一个世纪里，大规模的工业和航运事故以及不适当的废物处理做法使水生和陆地环境受到单体苯乙烯的严重污染。在工作场所，参与苯乙烯基材料配方和应用的个人面临高水平接触的最大风险。作为一种生化毒素，苯乙烯诱导铁和黄素依赖的单加氧酶的活性，催化环氧化和羟基化反应，产生强烷基化剂和肺刺激物，如苯乙烯氧化物和乙烯酚。我们研究的长期目标是阐明参与苯乙烯代谢途径的酶的结构和机制，并建立一个模型，以便更准确地评估与暴露相关的人类健康风险。我们的工作也将为其他代谢和解毒途径的研究提供一个框架，其中包括有毒或不稳定途径中间体的合成。苯乙烯代谢途径的酶——苯乙烯单加氧酶、苯乙烯氧化异构酶和苯乙醛脱氢酶已经克隆，并将通过机制和结构研究进行研究。苯乙烯单加氧酶将在我们的实验室进行功能表征，并由西北大学的Amy Rosenzweig博士的小组通过x射线晶体学进行结构表征。单次转化和稳态动力学研究将用于表征苯乙烯环氧化反应中涉及的中间体，并确定蛋白质-蛋白质相互作用在反应速率调节中的作用。将进行停止流动和快速淬火相结合的研究，以建立苯乙烯代谢中活性底物和辅酶运输的机制和效率。对苯乙烯单加氧酶的衍射质量晶体进行求解，并用于鉴定参与催化的活性位点结构。这项工作将导致对黄蛋白环氧化酶的第一个完整的结构和机制评价。阐明苯乙烯代谢过程中产生和穿梭有毒中间体的酶结构和机制是确定与人类和环境接触单体苯乙烯相关的健康风险的重要步骤。

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.