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

描述(申请人提供)：氧氯酸盐是人造杀生剂、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通过酶作用对这些污染物进行解毒，将它们的还原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-)，它本身是一种受EPA控制的化合物，广泛用于漂白和杀微生物剂。这些生物中的亚氯酸盐通过一种依赖于血红素的O-O键形成机制被亚氯酸盐歧化酶(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必需的反应，其中反应的特异性由蛋白质环境决定。拟议工作的第一个目标是确定结构-功能关系，允许来自模型高氯酸盐呼吸器的CLD以非凡的专一性催化亚氯酸盐的分解。在形成我们的假设时，我们利用了有文献记载的血红素过氧化物酶的结构-功能模型，这些模型被认为与CLD有共同的活性部位，但不具有催化或进一步的结构相似。用于实现这一目标的方法包括定点突变、稳态和快速动力学、共振拉曼光谱和X射线结晶学。第二个目标是阐明Cld在数百种细菌中的广泛作用，甚至是在已发现Cld同源物的古细菌中。有人提出，在高氯酸盐呼吸细菌中，由于高氯酸盐污染所施加的人为选择压力，亚氯酸盐错位是相对较新的进化。祖先的Cld基因产物在非高氯酸盐呼吸器中的功能完全未知，可能与绿泥石无关。这些生物体中的CLD有望发挥重要的、潜在的新型抗氧化剂作用。为了实现这一目标，将表达代表来自非呼吸器的两组CLD序列的CLD。将进行一系列化学和动力学实验，首先确定每种酶都能发挥的催化或传感器调节功能，然后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战略提供了必要的知识和材料，或者为CLD产生氧气的化学的生物技术应用(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：关于一种全新的由血红素催化的反应，以及关于一个广泛存在的、高度保守但尚未描述的微生物酶家族，该家族可能具有重要的抗氧化功能。最后，亚氯酸盐和相关化合物是杀微生物剂。因此，这项工作提供了一种新形式的进化的抗杀微生物剂耐药性的范例。与公共健康相关：氯氧酸盐是一种人造漂白剂类化学物质，最近已成为淡水的严重和普遍污染物。这项拟议的工作将定义某些微生物用来解毒氧氯酸盐的天然分子系统的关键特征，产生无害的氯离子和氧气气体。这项工作对于设计高效的氧氯酸盐生物修复策略以及其他产生O2的生物技术应用至关重要。

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. 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.