Cupin Dioxygenases: Role of Metal Center in Oxygen Activation

需氧生物已经进化到可以利用氧的氧化潜能来为自己的利益服务。虽然是一种强大的氧化剂，但二氧与其他分子的直接反应是缓慢的，自然界已经开发出降低这种动力学屏障的方法。这个氧活化的过程具有巨大的重要性，而理解这个活化完成的原理是至关重要的。最近介绍了一种研究氧活化的新工具，它分析了pH、粘度、温度和同位素组成对二氧与酶反应的二级速率常数的影响。这些研究已经提供了一些酶系统中氧活化速率决定步骤性质的有价值的信息。本项目描述了在cupin超家族的一组双加氧酶中，金属中心在氧和底物活化机制中的作用。初步研究选择了两种类型的酶系统：来自黄曲霉和枯草芽孢杆菌的含铜和含铁的黄酮醇2,3-双加氧酶，以及来自肺炎克雷伯菌的含镍和含铁的酸还原酮双加氧酶。选择这些酶是因为它们具有独特的机制，并且有可能为了解氧和底物活化的机制提供重要的见解。了解cupin蛋白超家族催化多样性的结构基础可以为破译新蛋白成员的功能提供基础。广泛影响：本项目结合生物化学、生物信息学、分子生物学、物理和合成有机化学的工具和技术，研究金属酶的作用机制。这项多学科研究为研究生和本科生的教育提供了一个极好的机会，在一个多元文化的研究环境中，这将促进少数民族学生的指导。

Aerobic organisms have evolved to use the oxidizing potential of oxygen to their benefit. Although a powerful oxidizing agent, the direct reaction of dioxygen with other molecules is slow and nature has developed ways to lower this kinetic barrier. This process of oxygen activation has tremendous importance and the understanding of the principles by which this activation is accomplished is crucial. A new tool for the study of oxygen activation was recently introduced and involves the analysis of the effects of pH, viscosity, temperature, and isotopic composition on the second order rate constant for the reaction of dioxygen with an enzyme. Such studies have already provided valuable information on the nature of the rate-determining steps in oxygen activation in a number of enzyme systems. This project describes efforts towards the elucidation of the role of the metal center in the mechanism of oxygen and substrate activation in a group of dioxygenases belonging to the cupin superfamily. Two types of enzyme systems have been chosen for initial studies: copper-and iron-containing flavonol 2,3-dioxygenases from Aspergillus flavus and Bacillus subtilis, and nickel- and iron-containing acireductone dioxygenases from Klebsiella pneumoniae. These enzymes were chosen for their unique mechanisms and the potential to provide important insights into the mechanism of oxygen and substrate activation. Understanding of the structural basis for the catalytic diversity observed in the cupin superfamily of proteins may provide the foundation for deciphering the function of new protein members.Broader Impact: This project incorporates tools and techniques of biochemistry, bioinformatics,molecular biology,physical and synthetic organic chemistry, in the study of the mechanism of metalloenzymes. This multidisciplinary research provides an excellent opportunity for the education of graduate and advanced undergraduate students in a multicultural research environment that will foster mentoring of minority students.