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

基本信息

批准号：
8766593
负责人：
Jennifer L DuBois
金额：
$ 34.44万
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 2015-09-04
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8766593
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

项目摘要

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）：草氯酸盐是人造杀生物剂、漂白剂和氧化剂，由于其广泛使用、高溶解度和毒性，目前已成为淡水的严重污染物。最近发现微生物可以通过酶解这些污染物的毒素，将污染物的减少与呼吸能量的产生结合起来。还原途径的最终产物是亚氯酸盐 (ClO2-)，它本身是 EPA 监管的化合物，也是广泛使用的漂白剂和杀菌剂。这些生物体中的亚氯酸盐被亚氯酸盐歧化酶 (Cld) 通过血红素依赖性 O-O 键形成机制转化为无害的 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需的反应，其中反应特异性由蛋白质环境决定。拟议工作的第一个目标是定义结构-功能关系，使来自高氯酸盐呼吸器模型的 Cld（芳香脱氯单胞菌）能够以非凡的特异性催化亚氯酸盐的分解。在形成我们的假设时，我们利用了有据可查的血红素过氧化物酶结构功能模型，预计它们与 Cld 共享活性位点，但不具有催化或进一步的结构相似性。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 CLD 在数百种细菌甚至古细菌中的更广泛作用，其中已发现 CLD 同源物。有人提出，亚氯酸根歧化是在高氯酸盐呼吸细菌中最近进化的，以响应高氯酸盐污染所施加的人为选择压力。祖先 cld 基因产物在非高氯酸盐呼吸器中的功能完全未知，并且可能与亚氯酸盐无关。 Cld 在这些生物体中预计将发挥重要且潜在新颖的抗氧化作用。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，首先确定每种酶所具有的催化或传感器调节功能，其次确定酶执行这些功能的效率。这项工作对健康的潜在影响有几个：首先，它为针对草氯酸盐的生物修复策略或 Cld 产生 O2 的化学物质的生物技术应用（例如，用于伤口愈合或水净化）提供了必要的知识和材料。其次，它提供了两种基本信息：关于一种全新的血红素催化反应，以及关于一种广泛存在、高度保守但尚未描述的可能具有重要抗氧化功能的微生物酶家族。最后，亚氯酸盐和相关化合物是杀菌剂。因此，这项工作为进化的抗微生物剂耐药性的新形式提供了范例。