Chlorite dismutase: a novel heme enzyme and its implications for human health

亚氯酸盐歧化酶：一种新型血红素酶及其对人类健康的影响

• 批准号: 8311778
• 负责人: Jennifer L DuBois
• 金额: $ 34.73万
• 依托单位: SRI INTERNATIONAL
• 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 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8311778
• 关键词: Active Sites; Address; Antioxidants; Archaea; Bacteria; Biochemical; Biochemistry; Biocide; Biology; Bioremediations; Biotechnology; Catalysis; Characteristics; Chemicals; Chemistry; Coupled; Coupling; Decontamination; Disinfection; Drug Metabolic Detoxication; Electrons; Engineering; Environment; Enzymatic Biochemistry; Enzymes; Evolution; Family; Fresh Water; Funding; Gases; Generations; Genes; Goals; Health; Heme; Homologous Gene; Human; In Situ; Kinetics; Knowledge; Metalloproteins; Methods; Microbe; Microbial Genome Sequencing; Modeling; Molecular; Necrosis; Operative Surgical Procedures; Organism; Orthologous Gene; Pathway interactions; Perchlorates; Peroxidases; Peroxonitrite; Play; Pollution; Proteins; Proteobacteria; Raman Spectrum Analysis; Reaction; Resistance; Respiration; Role; Salts; Series; Site-Directed Mutagenesis; Solubility; Specificity; Structure; Structure-Activity Relationship; System; Tissues; Toxic effect; Water; Water Pollutants; Work; Wound Healing; X-Ray Crystallography; anthropogenesis; base; chemical kinetics; chlorination; chlorite; heme a; interest; journal article; man; meetings; member; microbial; microbicide; novel; oxidation; pathogen; pressure; research study; respiratory; response; sensor; small molecule

DESCRIPTION (provided by applicant): The oxochlorates are man-made biocides, bleaches, and oxidizers that, because of their extensive use, high solubility, and toxicity, are now serious contaminants of fresh water. Microbes were recently discovered that can detoxify these contaminants enzymatically, coupling their reduction to the generation of respiratory energy. The end product of the reduction pathway is chlorite (ClO2-), itself an EPA-regulated compound and widely used bleach and microbicide. Chlorite in these organisms is converted by the enzyme chlorite dismutase (Cld) to harmless Cl- and O2 via a heme-dependent, O-O bond forming mechanism which we have recently begun to elucidate. Heme enzymes catalyze an array of biologically essential reactions, where reaction specificity is dictated by the protein environment. The first objective of the proposed work is to define the structure-function relationships that allow a Cld from a model perchlorate respirer, Dechloromonas aromatica, to catalyze the decomposition of chlorite with extraordinary specificity. In forming our hypotheses, we have drawn on well documented structure-function models for heme peroxidases, which are expected to share active site but not catalytic or further structural similarities with Cld. Methods to be used in pursuing this objective include site directed mutagenesis, steady state and rapid kinetics, resonance Raman spectroscopy, and X-ray crystallography. The second objective is to elucidate the broader role of Clds in the hundreds of bacteria and even archaea where cld homologs have been found. It has been proposed that chlorite dismutation evolved relatively recently, in perchlorate-respiring bacteria, in response to the anthropogenic selection pressure applied by perchlorate pollution. The function of the ancestral cld gene product in non-perchlorate respirers is completely unknown, and likely unrelated to chlorite. Cld in these organisms is expected to play an important and potentially novel antioxidant role. In pursuit of this objective, Clds representing the two groups of cld sequences from non-respirers will be expressed. A series of chemical and kinetic experiments will be carried out, first to define the catalytic or sensor-regulator functions that each is capable of, and second to determine the efficiency with which the enzymes carry out these functions. The potential health impacts of this work are several: first, it provides essential knowledge and materials for bioremediation strategies against oxochlorates, or for biotechnological applications of Cld's O2 generating chemistry (e.g., for wound healing or water decontamination). Second, it supplies two kinds of fundamental information: about an entirely novel heme-catalyzed reaction, and about a widespread, highly conserved, and yet un-described microbial enzyme family that likely has an important antioxidant function. Finally, chlorite and related compounds are microbicides. This work consequently offers a paradigm for a new form of evolved anti-microbicide resistance. PUBLIC HEALTH RELEVANCE: The oxochlorates are man-made, bleach-like chemicals that have recently become serious and widespread contaminants of fresh water. The proposed work will define critical features of the natural molecular systems used by certain microbes to detoxify oxochlorates, generating harmless Cl- and O2 gas. This work is essential for engineering efficient bioremediation strategies for the oxochlorates, as well as for other biotechnological applications of O2 generation.

描述（由申请人提供）：氧代氯酸盐是人造生物杀灭剂、漂白剂和氧化剂，由于其广泛使用、高溶解度和毒性，现在是淡水的严重污染物。最近发现的微生物可以酶解这些污染物，将它们的还原与呼吸能量的产生相结合。还原途径的最终产物是ClO 2-，它本身是EPA调节的化合物，广泛使用的漂白剂和杀微生物剂。这些生物中的亚氯酸盐通过血红素依赖的O-O键形成机制被酶亚氯酸盐歧化酶（Cld）转化为无害的Cl-和O2，我们最近开始阐明这一机制。血红素酶催化一系列生物学上必要的反应，其中反应特异性由蛋白质环境决定。建议的工作的第一个目标是定义的结构-功能的关系，允许Cld从模型高氯酸盐呼吸器，Dechloromonasaromatica，以催化分解具有非凡的特异性。在形成我们的假设，我们已经借鉴了有据可查的结构功能模型血红素过氧化物酶，预计共享活性位点，但没有催化或进一步的结构相似性Cld。用于实现这一目标的方法包括定点诱变、稳态和快速动力学、共振拉曼光谱和X射线晶体学。第二个目标是阐明更广泛的作用，Clds在数百种细菌，甚至古细菌，其中cld同系物已经found. It已提出，clad dismutation进化相对最近，在高氯酸盐呼吸细菌，在应对人为选择压力施加高氯酸盐污染。祖先cld基因产物在非高氯酸盐呼吸者中的功能完全未知，可能与高氯酸盐无关。Cld在这些生物体中有望发挥重要的和潜在的新的抗氧化作用。为了实现这一目标，将表达代表来自非呼吸者的两组cld序列的Cld。将进行一系列化学和动力学实验，首先确定每种酶的催化或传感调节功能，其次确定酶执行这些功能的效率。这项工作的潜在健康影响有几个：首先，它为针对氧氯酸盐的生物修复策略或Cld的O2生成化学的生物技术应用提供了基本知识和材料（例如，用于伤口愈合或水净化）。其次，它提供了两种基本信息：关于一个全新的血红素催化反应，以及关于一个广泛的，高度保守的，但未描述的微生物酶家族，可能具有重要的抗氧化功能。最后，甘草酸和相关化合物是杀微生物剂。因此，这项工作为一种新形式的进化抗杀微生物剂抗性提供了一个范例。公共卫生相关性：氯氧酸盐是一种人造的漂白剂，最近已经成为淡水的严重和广泛的污染物。拟议的工作将定义某些微生物用于解毒氧氯酸盐的天然分子系统的关键特征，产生无害的Cl-和O2气体。这项工作是必不可少的工程有效的生物修复策略的oxochlorates，以及其他生物技术应用的O2生成。