Understanding the novel reactivity of chlorite dismutases

了解亚氯酸盐歧化酶的新反应性

• 批准号: 8879585
• 负责人: Gudrun Susanne Lukat-Rodgers
• 金额: $ 33.5万
• 依托单位: NORTH DAKOTA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8879585
• 关键词: Active Sites; Address; Affinity; Bacteria; Bacterial Proteins; Binding; Cations; Characteristics; Chlorine; Complex; Cytochrome Peroxidase; Data; Decarboxylation; Distal; Drug Metabolic Detoxication; Drug resistance; Electron Spin Resonance Spectroscopy; Environment; Enzymes; Eukaryota; Evolution; Family; Fingerprint; Freezing; Generations; Goals; Gram-Positive Bacteria; Health care facility; Heme; Hemeproteins; Homologous Gene; Human; Hypochlorite; Isotope Labeling; Kinetics; Klebsiella pneumonia bacterium; Knowledge; Ligands; Measures; Metals; Methods; Modeling; NBL1 gene; Nitrates; Organism; Oxygen; Pathway interactions; Peracetic Acid; Perchlorates; Peroxonitrite; Plague; Poison; Porphyrins; Preparation; Production; Property; Protein Family; Proteins; Proteobacteria; Raman Spectrum Analysis; Reaction; Reporting; Respiration; Role; Sampling; Site; Staphylococcus aureus; System; Testing; Therapeutic; Work; antimicrobial; ascorbate; base; chlorite; coproporphyrinogen III; electronic structure; enzyme substrate complex; heme biosynthesis; insight; interest; member; novel; novel therapeutics; oxidation; pathogen; pathogenic bacteria; photosystem II; protein complex; protoporphyrin IX; public health relevance; rapid technique; resistant strain; respiratory

 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.

 描述（由申请人提供）：目前已知只有两种充分表征的酶系统催化O-O键的形成作为其主要功能。它们是在几种变形菌的高氯酸盐呼吸途径和光系统II的放氧复合物中发现的含血红素的磷酸二氢盐歧化酶（Cld）。Clds通过将其转化为O2和Cl-来降解有毒物质。Clds的O-O键形成反应的罕见性及其在各种生物医学和技术应用中根据需要解毒或产生O2的实用性导致了对这些酶的相当大的兴趣。事实证明，Clds包含一个庞大而广泛的酶家族，尽管它们具有共同的结构折叠，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起该家族的各种，在某些情况下是独特的功能。在此，我们拟研究该家族中三种具有代表性的Cld：芳香去氯单胞菌Cld（Dechloromonasaromatica Cld，DaCld），其在无氧呼吸过程中能高效地从厌氧微生物中产生O2，对高氯酸盐还原产物进行解毒;肺炎克雷伯氏菌Cld（Klebsiellapneumoniae，KpCld），其催化厌氧微生物分解反应的效率低于DaCld，其功能目前尚不清楚;和金黄色葡萄球菌Cld（SaCld），也称为HemQ，其不具有血红素分解活性，但对于血红素生物合成是必需的。除了了解活性中心环境变量如何指导三种Cld类型的反应性外，我们还希望深入了解1）一种新的反应机制， O2的产生; 2）Clds在革兰氏阴性菌如克雷伯氏菌中的可能作用。肺炎链球菌等重要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，其耐药菌株正在美国各地的医疗机构中流行。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，因此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标。具体而言，该项目的目标有三个方面：1）阐明DaCld/亚氯酸盐分解反应中O2释放的关键中间体的结构特征，2）检查KpCld与过氧化氢和过氧亚硝酸盐的酶反应性和反应中间体，以评估KpCld可能的解毒功能，3）通过确定SaCld（HemQ）与粪血红素的反应机制，确定其在血红素生物合成中的参数作用。这些目标将解决光谱（共振拉曼和瞬态吸收）和动力学（停止流动和冷冻淬火）的方法来确定原子的连接性和结构和电子性质的Cld反应中间体。这些研究支持了我们理解血红素环境如何指导酶功能的长期目标。