Structure and Mechanisms of Styrene Monooxygenase

苯乙烯单加氧酶的结构和机制

• 批准号: 7678363
• 负责人: George T. Gassner
• 金额: $ 23.03万
• 依托单位: SAN FRANCISCO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7678363
• 关键词: Accidents; Active Sites; Adhesives; Alkylating Agents; Amino Acids; Biochemical; Carcinogens; Catalysis; Coenzymes; Cytochromes; Drug Formulations; Drug Metabolic Detoxication; Electronics; Electrons; Elements; Ensure; Environment; Environmental Exposure; Environmental Hazards; Environmental Pollution; Enzymes; Evaluation; Exposure to; Fatty Acids; Flavins; Flavoproteins; Generations; Goals; Health; Human; Hydroxylation; Individual; Iron; Irritants; Isomerase; Kinetics; Laboratories; Lung; Maps; Metabolic; Metabolic Pathway; Metabolism; Mixed Function Oxygenases; Modeling; Nucleotides; Oxidoreductase; Oxygen; Pathway interactions; Phenols; Plant Resins; Plastics; Preparation; Production; Property; Reaction; Refuse Disposal; Research; Resources; Risk; Roentgen Rays; Role; Rubber; Series; Shipping; Ships; Societies; Staging; Structural Protein; Structure; System; Testing; Toxin; Transportation; Universities; Work; Workplace; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; activation product; base; chemical synthesis; directed evolution; electron density; enzyme mechanism; enzyme structure; epoxidase; high risk; instrumentation; microbial; microorganism; phenylacetic acid; protein protein interaction; reaction rate; styrene oxide

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.

描述（由申请人提供）：苯乙烯是各种塑料，橡胶和柜台粘合剂产品的重要组成部分。在过去的一个世纪中，大规模的工业和运输事故以及不适当的废物处理方式导致水生和陆地环境被单体苯乙烯污染。在工作场所，涉及苯乙烯基材料配方和应用的个人面临着高水平接触的最大风险。作为生化毒素，苯乙烯诱导了铁和黄素依赖性单加氧酶的活性，从而催化环氧化和羟基化反应可产生较强的烷基化剂，以及诸如苯乙烯氧化苯乙烯和乙烯基酚类等肺刺激剂。我们研究的长期目标是阐明从事苯乙烯代谢途径的酶的结构和机制，并建立一个模型，以更准确地评估与暴露相关的人类健康风险。我们的工作还将为研究其他代谢和排毒途径的研究提供一个框架，其中包括综合有毒或不稳定的途径中间体。苯乙烯代谢途径，苯乙烯单加氧酶，苯乙烯异构酶和苯乙醛脱氢酶的酶已被克隆，并将通过机械和结构研究来研究。苯乙烯单加氧酶将在我们的实验室中进行功能表征，并在结构上通过X射线晶体学表征了西北大学的艾米·罗森茨威格（Amy Rosenzweig）小组的X射线晶体学表征。单转化和稳态动力学研究将用于表征与苯乙烯环氧反应有关的中间体，并确定蛋白质 - 蛋白质相互作用在反应速率调节中的作用。将进行停止流量和快速淬火研究的结合，以确定苯乙烯代谢中反应性底物和辅酶转运的机理和效率。苯乙烯单加氧酶的衍射质量晶体将得到解决，并用于识别参与催化的活性位点结构。这项工作将导致黄酮蛋白环氧酶的首次完整结构和机械评估。在苯乙烯代谢过程中阐明从事有毒中间体的产生和穿梭的酶结构和机制是确定与人类和环境暴露于单体型苯乙烯相关的健康风险的重要步骤。