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

基本信息

批准号：
9137697
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
项目类别：
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/9137697
关键词：
Accounting Address Aerobic Air Ally Anthrax disease Antibiotic Resistance Archaea Bacteria Basic Science Binding Biochemical Biochemical Pathway Biochemistry Biology Carbon Carbon Dioxide Catalysis Cell Respiration Cell physiology Cells Chemistry 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 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 tool

项目摘要

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）：血红素对于有氧生命和细胞呼吸至关重要。相比之下，原核细胞血红素生物合成的途径最近却很难描述。革兰氏阳性菌的血红素生物合成填补了所有剩余的空白。革兰氏阳性菌是一组生物体，其中包括许多威胁公共健康和健康的重要病原体。生物防御，例如耐甲氧西林金黄色葡萄球菌（MRSA）、结核病、炭疽病和鼠疫的病原体。该途径在最后三个步骤中与经典途径不同，最大的不同在于最后一步是由已知酶催化的双重氧化脱羧。 HemQs：亚氯酸盐歧化酶 (Clds) 的一种新亚型，后者是对亚氯酸盐最终产物进行解毒的血红素酶。这项研究的初始阶段对高氯酸盐呼吸器和非呼吸病原体产生 O2 的结构、机制和生物学进行了严格的描述。见解以及通过 Clds 工作组建的科学团队，该提案旨在提供个人层面上 HemQ 功能的描述作为初步工作，金黄色葡萄球菌的 hemQ 菌株已被生成，并被证明是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耐药性相关的表型。来自金黄色葡萄球菌的 HemQ 酶已被证明可以使粪血红素 III 的四个丙酸侧链中的两个氧化脱羧，该反应严格依赖于 H2O2。重点关注金黄色葡萄球菌系统，目标 1 是了解 HemQ 如何结合并激活 coproheme 进行氧化脱羧，生成 SaHemQ 与其底物 (coproheme III)、中间体 (harderoheme) 和产物 (heme b) 复合物的结构和能量模型目标 2 是测试 HemQ 反应的机制，其中粪血红素既是过氧化反应的底物又是辅助因子。时间分辨和动力学同位素方法将用于检验一系列涉及铁-过氧化氢中间体和分子内氢原子转移的假设，最后，目标 3 使用遗传、细胞和生化方法来了解 HemQ 在我们期望完成拟议的工作，以定义一条途径的最终步骤，该途径对于有氧生命绝对重要，对于致病菌的强劲生长至关重要，并且在临床上与细胞和进化的发展相关。持久性和抗生素耐药性。