Cupin Dioxygenases: Role of Metal Center in Oxygen Activation

好氧生物已经进化到利用氧气的氧化潜力来造福于它们。尽管氧气是一种强大的氧化剂，但它与其他分子的直接反应是缓慢的，大自然已经找到了降低这一动力学障碍的方法。氧活化的这个过程非常重要，而理解完成这一活化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，它涉及到分析pH、粘度、温度和同位素组成对氧气与酶反应的二级速率常数的影响。这类研究已经为一些酶系统中氧激活的速率决定步骤的性质提供了宝贵的信息。本项目致力于阐明金属中心在Cupin超家族中的一组双加氧酶中氧和底物激活机制中的作用。选择了两种类型的酶系统进行初步研究：黄曲霉和枯草芽孢杆菌的含铜和铁的黄酮醇2，3-双加氧酶和肺炎克雷伯氏菌的含镍和含铁的酸性还原内酯双加氧酶。这些酶之所以被选中，是因为它们具有独特的机制，并有可能为氧和底物激活的机制提供重要的见解。了解在Cupin蛋白质超家族中观察到的催化多样性的结构基础，可能为破译新的蛋白质成员的功能提供基础。广泛的影响：这个项目结合了生物化学、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，研究金属酶的机制。这一多学科研究为在多元文化研究环境中培养研究生和高级本科生提供了一个极好的机会，这将促进对少数族裔学生的指导。

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.