Understanding the novel reactivity of chlorite dismutases

 描述(由申请人提供)：目前已知只有两个特征良好的酶系统催化O-O键的形成作为它们的主要功能。它们是在几种变形杆菌的高氯酸盐呼吸途径中发现的含血红素的亚氯酸盐歧化酶(Cld)和光系统II的放氧复合体。Clds通过将有毒的亚氯酸盐转化为O2和Cl2-来降解有毒的亚氯酸盐。由于ClDS的O-O成键反应的稀有性，以及它们在各种生物医学和技术应用中对亚氯酸盐的解毒或根据需要产生O2的有效性，导致了对这些酶的极大兴趣。事实证明，ClD由一个庞大而广泛的酶家族组成，尽管它们有共同的结构折叠，但具有不同的功能。我们的长期目标之一是了解血红素-蛋白质相互作用中的细微差异如何引发这个家族的各种功能，在某些情况下还具有独特的功能。在这里，我们建议研究该家族中三种具有代表性的Clds：芳香地氯单胞菌(DaCld)，它能从亚氯酸盐中产生氧气，在厌氧呼吸过程中对高氯酸盐还原产物进行解毒；肺炎克雷伯菌(Klebsiella)，其催化亚氯酸盐分解反应的效率低于DaCld，其功能目前尚不清楚；以及金黄色葡萄球菌(SaCld)，也被称为hemQ，它没有亚氯酸盐分解活性，但对血红素的生物合成是必不可少的。除了了解活性中心环境变量如何指导三种类型的反应外，我们还希望深入了解1)一种新的机制 第二，Clds在肺炎克雷伯菌等革兰氏阴性病原体中的可能作用(S)，以及3)金黄色葡萄球菌等关键革兰氏阳性病原体中血红素生物合成的新途径，金黄色葡萄球菌等耐药菌株正困扰着美国各地的医疗机构。由于在人类中没有发现来自革兰氏阳性细菌的CLD家族成员，一旦了解其功能的机制方面，CLD有望成为抗菌治疗的尚未开发的靶点。具体地说，该项目的目标有三个：1)阐明在大氯酸盐/亚氯酸盐分解反应中关键的放氧中间体的结构特征；2)检测KpCld与亚氯酸盐和过氧亚硝酸盐的酶反应活性和反应中间产物，以评估KpCld可能的解毒功能(S)；以及3)通过确定其与辅位的反应机制，确定其在血红素生物合成中的参数作用。这些目标将通过光谱(共振拉曼和瞬时吸收)和动力学(停流和冻结-猝灭)方法来实现，以确定CLD反应中间产物的原子连接性和结构和电子性质。这些研究支持了我们的长期目标，即了解血红素环境如何引导酶功能。

 DESCRIPTION (provided by applicant): Currently only two well characterized enzymatic systems are known to catalyze the formation of an O-O bond as their primary function. They are the heme-containing chlorite dismutases (Cld) found in the perchlorate respiratory pathway of several Proteobacteria and the oxygen-evolving complex of photosystem II. Clds degrade toxic chlorite by converting it to O2 and Cl-. The rarity of the O-O bond-forming reaction of Clds and their utility to detoxify chlorite or to produce O2 on demand in a variety of biomedical and technical applications resulted in considerable interest in these enzymes. Turns out, Clds comprise a large, widespread family of enzymes that, despite their common structural fold, have varied functions. One of our long term goals is to understand how subtle differences in the heme-protein interactions elicit the varied, and in some cases unique, functions of this family. Here we propose to study representative Clds from three types within the family: Dechloromonas aromatica Cld (DaCld) which produces O2 from chlorite with tremendous efficiency for detoxification of perchlorate reduction products during anaerobic respiration; Klebsiella pneumoniae Cld (KpCld) which catalyzes the chlorite decomposition reaction less efficiently than DaCld, and whose function is currently unknown; and Staphylococcus aureus Cld (SaCld) also known as HemQ, which has no chlorite decomposing activity, but is essential for heme biosynthesis. In addition to understanding of how the active site environment variables direct the reactivities the three Cld types, we expect to gain insight into 1) a novel mechanism of O2 production, 2) the possible role(s) of Clds in Gram-negative pathogens like K. pneumoniae and 3) a new pathway in heme biosynthesis in critically important Gram-positive pathogens like S. aureus, whose drug-resistant strains are plaguing healthcare facilities throughout the US. As no members of the Cld family from Gram-positive bacteria are found in humans, Cld holds promise as a yet unexploited target for antimicrobial therapeutics, once the mechanistic aspects of their functions are understood. Specifically, the aims of the project are threefold: 1) elucidat structural characteristics of intermediates key to O2 evolution in the DaCld/chlorite-decomposing reaction, 2) examine enzyme reactivity and reaction intermediates of KpCld with chlorite and peroxynitrite to assess possible detoxification function(s) of KpCld, and 3) parameterize role of SaCld (HemQ) in heme biosynthesis by determining its reaction mechanism with coproheme. These aims will be addressed with spectroscopic (resonance Raman and transient absorbance) and kinetic (stopped flow and freeze-quench) approaches to determining atom connectivities and structures and electronic properties of Cld reaction intermediates. These studies support our long term goal of understanding how heme environment directs enzyme function.