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为自己谋利。虽然是一种强氧化剂，但分子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障碍的方法。氧气活化的过程非常重要，理解实现这种活化的原理至关重要。最近介绍了一种研究氧活化的新工具，涉及分析pH、粘度、温度和同位素组成对双氧与酶反应的二级速率常数的影响。这些研究已经提供了有价值的信息的性质，在氧活化的速率决定步骤在一些酶系统。本计画描述了一组属于cupin超家族的双加氧酶中金属中心在氧和底物活化机制中的作用。已选择两种类型的酶系统进行初步研究：含铜和铁的黄曲霉和枯草芽孢杆菌的黄酮醇2，3-双加氧酶，和含镍和铁的酸还原酮双加氧酶从肺炎克雷伯氏菌。选择这些酶是因为它们独特的机制以及为氧气和底物活化机制提供重要见解的潜力。理解的催化多样性的结构基础观察cupin超家族的蛋白质可能提供的基础破译新的蛋白质members.Broader影响：本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，在研究的机制的金属酶。这种多学科的研究提供了一个很好的机会，研究生和高级本科生的教育，在多元文化的研究环境，将促进少数民族学生的辅导。

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.