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

这个项目是由分子和细胞生物科学部的分子生物物理组和化学部的生命过程化学计划共同资助的。这个项目研究了一种处于病原微生物和它们的植物/动物宿主之间的化学战中心的酶(KatG)。宿主产生过氧化氢作为复杂的微生物杀死系统的一部分，许多病原体产生这种酶来防御自己。产生KatG的病原体从稻瘟菌到结核病，前者每年造成数十亿美元的作物损失，后者每年导致数百万人死亡。显然，了解KatG是如何工作的很重要。这些知识可能会为未来的干预提供基础，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不足群体的高危学生的成功率，最大限度地减少他们在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，这项研究有助于为阿拉巴马州的囚犯创造新的STEM学习机会，因为它提供的材料能够激发囚犯的兴趣，并使他们未来参与STEM学习。分子内电子转移是KatG催化所有方面的中心，包括其蛋氨酸-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并未得到很好的定义。为了解决这些未知问题，本项目分为两个部分。第一个评估KatG已经包含其myw辅因子，第二个评估在辅因子形成之前的KatG。前者将阐明活性催化所需的中间体以及那些导致失活的中间体，而后者将阐述导致和削弱新的myw辅因子合成的中间体。在这两种情况下，将探讨KatG蛋白自身控制电子转移途径的机制，以及外源电子供体恢复不活跃中间体的能力。因此，从化学猝灭多肽图谱到冷冻猝灭穆斯堡尔谱学，一系列配对的动力学和光谱方法将应用于针对近端色氨酸和/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.