Conduits and Control of KatG Intramolecular Electron Transfer: Formation and Operation of a Novel Cofactor

本项目由分子和细胞生物科学部的分子生物物理学组和化学部的生命过程化学项目共同资助。本项目研究处于病原微生物与其植物/动物宿主之间肆虐的化学战中心的酶（KatG）。病原体产生过氧化氢作为复杂的微生物杀灭系统的一部分，许多病原体产生这种酶来保护自己。产生KatG的病原体包括稻瘟病真菌，每年造成数十亿美元的作物损失，以及每年导致数百万人死亡的结核病。很明显，了解KatG是如何工作的是很重要的。这些知识可能为未来的干预措施提供基础，使这些病原体更容易受到宿主自身防御的影响。该项目为研究生和本科生提供研究培训。最重要的是，它有助于提高来自代表性不足群体的高风险学生的成功率，最大限度地减少STEM专业的流失，并最大限度地提高他们完成学位的可能性。最后，这项研究有助于产生新的干学习机会，为亚拉巴马囚犯贡献材料，激发兴趣，从事未来参与干学习的囚犯。分子内电子转移是KatG催化的所有方面的核心，包括其甲硫氨酸-酪氨酸-色氨酸（MYW）辅因子的形成、辅因子用于催化、非活性中间体的积累以及这些中间体通过外源电子供体恢复活性。然而，这些过程的中间体及其控制机制的定义很差。为了解决这些未知数，这个项目分为两个部分。第一个评估KatG已经包含其MYW辅因子，第二个评估KatG辅因子形成之前。前者将阐明活性催化所必需的中间体以及导致失活的中间体，而后者将解决导致和减损从头MYW辅因子合成的中间体。在这两种情况下，KatG蛋白本身控制电子转移途径的机制将得到解决，外源电子供体恢复非活性中间体的能力也将得到解决。因此，一套广泛的配对动力学和光谱方法，从化学淬灭肽映射到冷冻淬灭穆斯堡尔谱，将应用于靶向近端色氨酸和/或精氨酸开关的KatG变体。

This project is jointly funded by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences and the Chemistry of Life Processes Program in the Division of Chemistry.This project investigates an enzyme (KatG) at the center of the chemical warfare that rages between pathogenic microorganisms and their plant/animal hosts. Hosts produce hydrogen peroxide as part of an elaborate microbial killing system, and many pathogens produce this enzyme to defend themselves. KatG-producing pathogens range from the rice-blast fungus, which annually costs billions of dollars in crop losses, to tuberculosis, which kills millions of people each year. Clearly, it is important to understand how KatG works. Such knowledge may provide a foundation for future interventions that make these pathogens more susceptible to their hosts' own defenses. This project provides research training for graduate and undergraduate students. Most importantly, it contributes to increasing success of at-risk students from underrepresented groups, minimizing their attrition from STEM majors and maximizing the likelihood they will complete their degrees. Finally, this research helps generate new STEM learning opportunities for Alabama prisoners by contributing material that excites interest and engages future participation of prisoners in STEM learning. Intramolecular electron transfer is central to all aspects of KatG catalysis, including formation of its methionine-tyrosine-tryptophan (MYW) cofactor, the use of the cofactor for catalysis, the accumulation of inactive intermediates, and return of those intermediates to activity by exogenous electron donors. However, the intermediates of these processes and their mechanisms of control are poorly defined. To address these unknowns, this project is divided into two parts. The first evaluates KatG already containing its MYW cofactor, and the second evaluates KatG prior to cofactor formation. The former will elucidate the intermediates necessary for active catalysis as well as those that lead to inactivation, whereas the latter will address the intermediates that lead to and detract from de novo MYW cofactor synthesis. In both cases, the mechanisms by which the KatG protein itself controls the routes of electron transfer will be addressed, as will the ability of exogenous electron donors to restore inactive intermediates. As such, a broad suite of paired kinetic and spectroscopic methods, from chemical-quench peptide mapping to freeze-quench Mossbauer spectroscopy, will be applied to KatG variants targeting the proximal tryptophan and/or the arginine switch.