Structure and Mechanisms of Styrene Monooxygenase

描述(由申请人提供)：苯乙烯是各种塑料、橡胶和场外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导致水和陆地环境受到单体苯乙烯的严重污染。在工作场所，参与以苯乙烯为基础的材料的配方和应用的个人处于高水平暴露的最大风险中。作为一种生化毒素，苯乙烯诱导铁和黄素依赖的单加氧酶活性，催化环氧化和羟基化反应产生强烷基化试剂和肺刺激物，如环氧苯乙烷和乙烯基酚。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，允许更准确地评估与暴露相关的人类健康风险。我们的工作还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、苯氧基异构酶和苯乙醛脱氢酶已被克隆，并将通过机理和结构研究进行研究。西北大学艾米·罗森茨韦格博士的团队将在我们的实验室中对苯乙烯单加氧酶进行功能表征，并通过X射线结晶学对其结构进行表征。单周转和稳态动力学研究将被用来表征参与苯乙烯环氧化反应的中间体，并确定蛋白质-蛋白质相互作用在调节反应速度中的作用。将进行停流和快速猝灭相结合的研究，以确定反应底物和辅酶在苯乙烯代谢中的转运机制和效率。将对具有衍射性的苯乙烯单加氧酶晶体进行解析，并用于鉴定催化作用下的活性中心结构。这项工作将导致对黄素蛋白环氧酶的第一个完整的结构和机制的评估。阐明在苯乙烯代谢过程中参与有毒中间体的产生和穿梭的酶结构和机制是识别与单体苯乙烯接触的人类和环境相关的健康风险的关键步骤。

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.