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）：目前已知只有两种特征明确的酶系统以催化 O-O 键的形成为主要功能，它们是在几种变形菌和细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）。光系统 II 的释氧复合物通过将有毒的亚氯酸盐转化为 O2 和 Cl- 来降解 O-O 的稀有性。 Clds 的成键反应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 的用途引起了人们对这些酶的极大兴趣。事实证明，Clds 包含一个大而广泛的酶家族，尽管它们具有多种性质。共同的结构折叠，具有不同的功能，我们的长期目标之一是了解血红素-蛋白质相互作用的微妙差异如何引发该家族的不同的、在某些情况下独特的功能。该科内的类型：芳香脱氯单胞菌 Cld (DaCld)，在无氧呼吸过程中以亚氯酸盐产生 O2，对高氯酸盐还原产物的解毒效率极高；肺炎克雷伯菌 Cld (KpCld)，其催化亚氯酸盐分解反应的效率低于 DaCld，其功能是目前未知；以及金黄色葡萄球菌 Cld (SaCld)，也称为 HemQ，它具有没有亚氯酸根分解活性，但对于血红素生物合成至关重要 除了了解活性位点环境变量如何指导三种 Cld 类型的反应之外，我们还希望深入了解 1) 的新机制。 O2 的产生，2）Clds 在革兰氏阴性病原体（如肺炎克雷伯菌）中的可能作用，3）在极其重要的革兰氏阳性病原体（如金黄色葡萄球菌）中血红素生物合成的新途径，其耐药菌株正在困扰由于在人类中没有发现革兰氏阳性菌的 Cld 家族成员，因此一旦机制确定，Cld 有望成为抗菌治疗的尚未开发的靶点。具体来说，该项目的目标有三个：1）阐明 DaCld/亚氯酸盐分解反应中 O2 演化的关键中间体的结构特征，2）检查 KpCld 与亚氯酸盐的酶反应性和反应中间体。过氧亚硝酸盐来评估 KpCld 可能的解毒功能，以及 3) 通过确定 SaCld (HemQ) 在血红素生物合成中的作用参数化这些目标将通过光谱（共振拉曼和瞬态吸光度）和动力学（停止流动和冷冻猝灭）方法来确定 Cld 反应中间体的原子连接性和结构和电子特性。了解血红素环境如何指导酶功能的目标。