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: 8634171
• 负责人: Jennifer L DuBois
• 金额: $ 1.62万
• 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/8634171
• 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.

描述（由申请人提供）：含氧氯酸盐是人为的杀菌剂，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。最近发现微生物可以通过酶促排毒这些污染物，并将其还原与呼吸能的产生结合。还原途径的最终产物是氯酸盐（clo2-），它本身是EPA调节的化合物，并且广泛使用的漂白剂和杀菌剂。这些生物中的氯酸盐通过酶的亚氯酸盐歧地（CLD）转化为无害的Cl-和O2，通过血红素依赖性的O-O键形成机制，我们最近开始阐明。血红素酶催化一系列生物学上的基本反应，其中反应特异性由蛋白质环境决定。拟议的工作的第一个目标是定义结构功能关系，该关系允许模型的高氯酸盐呼吸器Dechloromonas芳香族A型cld，以极其特异性催化氯酸盐的分解。在形成我们的假设时，我们借鉴了有据可查的血红素过氧化物酶的结构功能模型，这些模型有望与CLD共享活跃的位点，但不能与CLD共享催化或进一步的结构相似性。用于追求此目标的方法包括位点定向诱变，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CLD在发现CLD同源物的数百种细菌甚至古细菌中的广泛作用。已经提出，在高氯酸盐呼吸的细菌中，氯酸盐的抑制作用是相对演变的，这是对高氯酸盐污染施加的人为选择压力的响应。祖先CLD基因产物在非渗透呼吸器中的功能是完全未知的，可能与氯酸盐无关。这些生物中的CLD有望发挥重要且潜在的新型抗氧化作用。为了追求这一目标，将表达代表非抗议者CLD序列的CLD。将进行一系列化学和动力学实验，首先定义每个化学实验，以定义每个化学实验的功能，其次是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种：首先，它为针对氧化氯酸盐的生物修复策略或CLD的O2产生化学的生物技术应用提供了必不可少的知识和材料（例如，用于伤口愈合或水的净化）。其次，它提供了两种基本信息：关于一种完全新颖的血红素催化反应，以及一个可能具有重要的抗氧化功能的广泛，高度保守但未描述的微生物酶家族。最后，亚氯酸盐和相关化合物是菌皮。因此，这项工作提供了一种新形式的抗霉素耐药性的范式。