Understanding the diverse biochemistry of the chlorite dismutase family: from O2 to heme

了解亚氯酸盐歧化酶家族的多样化生物化学：从 O2 到血红素

• 批准号: 9332429
• 负责人: Jennifer L DuBois
• 金额: $ 29.06万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9332429
• 关键词: Address; Aerobic; Air; Anthrax disease; Antibiotic Resistance; Archaea; Bacteria; Basic Science; Binding; Biochemical; Biochemical Pathway; Biochemistry; Biology; Carbon; Carbon Dioxide; Catalysis; Cell Respiration; Cell physiology; Cells; Chemistry; Clinical; Complex; Computing Methodologies; Data; Decarboxylation; Development; Disease; Electrons; Environment; Enzymes; Eukaryota; Eukaryotic Cell; Evolution; Family; Genes; Genetic; Goals; Gram-Positive Bacteria; Growth; Health; Heme; Hydrogen; Hydrogen Peroxide; Individual; Infection; Informatics; Isotopes; Kinetics; Life; Link; Listeriosis; Metabolism; Metals; Methods; Mission; Modeling; Molecular Chaperones; Movement; National Institute of General Medical Sciences; Nature; Organism; PPIX; Pathogenicity; Pathway interactions; Perchlorates; Peroxides; Phase; Phenotype; Plague; Porphyrins; Positioning Attribute; Propionates; Protein Family; Proteins; Protons; Public Health; Reaction; Recombinants; Research; Respiration; Series; Side; Staphylococcus aureus; Structure; System; Testing; Time; Tuberculosis; Variant; Virulence; Work; abstracting; base; biodefense; chlorite; cofactor; coproporphyrinogen oxidase; enzyme substrate; ferrochelatase; ferryl iron; heme a; heme biosynthesis; innovation; insight; methicillin resistant Staphylococcus aureus; microbial; novel; pathogen; peroxidation; protonation; protoporphyrin IX; public health relevance; tool

 DESCRIPTION (provided by applicant): Heme is essential for aerobic life and cellular respiration. The pathway by which eukaryotic cells make heme has been known for some time. Prokaryotic heme biosynthesis, by contrast, has been harder to describe. Recently, a pathway for heme biosynthesis that fills all the remaining gaps has been proposed for Gram- positive bacteria. This is a group of organisms that includes numerous important pathogens that are threats to public health and biodefense, such as the causative agents of MRSA, TB, anthrax, and plague. The pathway differs from the canonical one in its final three steps, with the greatest departure at its terminus. The last step is a double oxidative decarboxylation catalyzed by enzymes known as HemQs: a novel subtype of chlorite dismutases (Clds). The latter are heme enzymes that detoxify the chlorite end product of perchlorate respiration, converting it to Cl- to O2. The initial phase of this research resulted in a rigorous description of the structure, mechanism, and biology of O2-generating Clds from both perchlorate respirers and non-respiring pathogens. Leveraging the tools, insights, and scientific team assembled via work on Clds, this proposal aims at providing a description of HemQ function at the level of the individual molecule and extending to the cellular context. As preliminary work, a hemQ strain of Staphylococcus aureus has been generated and shown to be a heme auxotroph and small colony variant (SCV): a phenotype associated with intracellular persistence and antibiotic resistance. In tandem, the HemQ enzyme from S. aureus has been shown to oxidatively decarboxylate two of the four propionate side chains of coproheme III, in a reaction that depends strictly H2O2. Focusing on the S. aureus system, Aim 1 is to understand how HemQ binds and activates coproheme toward oxidative decarboxylation, producing structural and energetic models of SaHemQ in complex with its substrate (coproheme III), intermediate (harderoheme) and product (heme b). Aim 2 is to test a mechanism for HemQ's reaction, in which coproheme is both substrate and cofactor in the peroxidation. Time-resolved and kinetic isotope methods will be used to examine a series of hypotheses invoking a ferric-hydroperoxy intermediate and intramolecular hydrogen atom transfer. Finally, aim 3 uses genetic, cell-based, and biochemical methods to understand HemQ's function in the context of the cell and evolution. We expect completion of the proposed work to define the ultimate step of a pathway that is absolutely fundamental to aerobic life, essential for robust pathogenic growth, and clinically connected to the development of persistence and antibiotic resistance.

 描述（由申请人提供）：血红素是有氧生命和细胞呼吸所必需的。真核细胞制造血红素的途径已经知道一段时间了。相比之下，原血红素的生物合成则更难描述。最近，已经提出了用于填补革兰氏阳性细菌的所有剩余缺口的血红素生物合成途径。这是一组生物体，包括许多对公共卫生和生物防御构成威胁的重要病原体，如MRSA，TB，炭疽和鼠疫的病原体。这条道路在最后三个步骤与经典的道路不同，最大的偏离在它的终点。最后一步是由称为HemQs的酶催化的双重氧化脱羧作用：HemQs是一种新的α-dismutases（Clds）亚型。后者是血红素酶，可以将高氯酸盐呼吸的最终产物解毒，将其转化为Cl-和O2。这项研究的初始阶段导致了严格的描述的结构，机制和生物学的O2生成Clds从高氯酸盐呼吸和非呼吸病原体。利用通过Clds工作聚集的工具，见解和科学团队，该提案旨在提供个人层面的HemQ功能描述 分子并延伸到细胞环境。作为初步工作，已经产生了金黄色葡萄球菌的hemQ菌株，并且显示为血红素营养缺陷型和小菌落变体（SCV）：与细胞内持久性和抗生素抗性相关的表型。串联，来自S.金黄色葡萄球菌已显示在严格依赖于H2 O2的反应中使粪血红素III的四个丙酸酯侧链中的两个氧化脱羧。专注于S。金黄色葡萄球菌系统，目的1是了解HemQ如何结合并激活粪血红素进行氧化脱羧，产生SaHemQ与其底物（粪血红素III）、中间体（硬血红素）和产物（血红素B）复合的结构和能量模型。目的二是探讨血红素Q的反应机理，其中粪血红素既是过氧化反应的底物，又是过氧化反应的辅因子。时间分辨和动力学同位素的方法将被用来检查一系列的假设调用一个铁氢过氧中间体和分子内氢原子转移。最后，目标3使用遗传，基于细胞和生物化学的方法来理解HemQ在细胞和进化背景下的功能。我们希望完成拟议的工作，以确定对有氧生活绝对重要的途径的最终步骤，对强大的病原体生长至关重要，并在临床上与持久性和抗生素耐药性的发展有关。