Studies of Tetrachloro-1,4-Hydroquinone Dehalogenase

Copley四氯-1,4-对苯二酚脱卤酶在谷胱甘肽依赖反应中催化四氯-1,4-对苯二酚（TCHQ）和三氯-1,4-对苯二酚（TriCHQ）的还原脱卤，该酶在土壤微生物Flavobacterium sp.菌株ATCC 39723和Sphingomonas sp.菌株RA-2中降解五氯酚的途径中被发现。催化还原脱卤反应的酶，通常由沉积物和土壤中的厌氧微生物进行。由于这些反应通常非常缓慢，高度卤化的化合物可能在环境中持续存在数十年。尽管还原脱卤很重要，但催化这些反应的酶的机制或结构信息尚不清楚。唯一被纯化的还原芳基脱卤酶是TCHQ脱卤酶。这一提议描述了这种新酶的第一个机制实验。本提案中描述的具体工作目标是：1)确定酶上的金属离子、辅因子或半胱氨酸残基是否参与还原性等价物从谷胱甘肽转移到TCHQ；2)确定氯和羟基取代基对催化的影响；3)测量动力学同位素效应，以确定氢向芳环的转移或碳氯键的裂解是否参与了速率决定步骤。这些实验将使我们能够提出TCHQ脱卤酶的机制，以便在未来进行更详细的研究。生物降解领域的一个关键问题是微生物如何进化出降解五氯酚等化合物的能力，这些化合物直到最近才大量存在于环境中。对参与卤代芳香族化合物降解的酶的进化起源问题的一种方法是比较一些微生物中的酶和相应的基因序列。第二部分重点比较黄杆菌ATCC 39723和鞘氨单胞菌RA-2中TCHQ脱卤酶及其基因。在过去的五十年中，由于生产过程、无意的泄漏、不良的处理方法和杀虫剂的使用增加，大量的氯化芳香族化合物被引入环境中。许多氯化芳香族化合物被自然存在的微生物降解得非常缓慢。抗生物降解的程度通常随着氯取代基数量的增加而增加。因此，多氯联苯、六氯苯和五氯酚等高度氯化的化合物往往会在环境中持续存在。高氯化芳香族化合物的生物降解始于脱氯反应，用氢原子取代氯原子。一旦从环中除去足够数量的氯原子，这些化合物就可以相对较快地降解。尽管这种类型的脱氯反应很重要，但没有关于催化这些反应的酶的机制或结构信息。本提案的第一部分描述了这种酶的第一个机械实验。生物降解领域的一个关键问题是微生物如何进化出降解五氯酚等化合物的能力，这些化合物直到最近才大量存在于环境中。解决这个问题的一种方法是比较一些微生物中的酶和相应的基因序列。第二部分重点比较了黄杆菌ATCC 39723和Spingomonas sp. RA-2两种不同土壤微生物的脱卤酶活性。* * *

9406909 Copley Tetrachloro-1,4-hydroquinone dehalogenase catalyzes the reductive dehalogenation of tetrachloro-1,4-hydroquinone (TCHQ) and trichloro-1,4-hydroquinone (TriCHQ) in a glutathione-dependent reaction this enzyme is found in the pathway for pentachlorophenol degradation in the soil microorganisms Flavobacterium sp. strain ATCC 39723 and Sphingomonas sp. strain RA-2. Enzymes that catalyze reductive dehalogenation reactions, usually carried out by anaerobic microorganisms in sediments and soils. Since these reactions are generally very slow, highly halogenated compounds may persist in the environment for decades. Despite the importance of reductive e dehalogenatiion, no mechanistic or structural information is available about the enzymes that catalyze these reactions. the only reductive aryl dehalogenasae that has been purified is TCHQ dehalogenase. This proposal describes the first mechanistic experiments of this novel enzyme. The specific goals of the work described in this proposal are 1) to determine whether metal ions, cofactors, or cysteine residues on the enzyme are involved in transfer of reducing equivalents from glutathione to TCHQ;2) to determine the effect of the chlorine and hydroxyl substituents on catalysis; and 3) to measure kinetic isotope effects to determine whether transfer of hydrogen to the aromatic ring or carbon-chlorine bond cleavage are involved in the rate- determining step. These experiments will allow us to propose a mechanism for TCHQ dehalogenase for more detailed studies in the future. A critical question in the field of biodegradation is how microorganisms have evolved the ability to degrade compounds such as pentachlorophenol which were not present in large quantities in the environment until relatively recently. One approach to the question of he evolutionary origin of enzymes involved in degradation of halogenated aromatic compounds is to compare the enzymes and corresponding gene sequences in a number of micro organisms. The second part of this proposal focuses on a comparison of the TCHQ dehalogenase enzymes and the corresponding genes in Flavobacterium sp. strain ATCC 39723 and Sphingomonas sp. strain RA-2. %%% During the last fifty years, large quantities of chlorinated aromatic compounds have been introduced into the environment as a result of manufacturing processes, inadvertent spills, poor disposal practices and increased use of pesticides. Many chlorinated aromatic compounds are degraded only very slowly by naturally-occurring microorganisms. The degree of resistant to biodegradation usually increases with the number of chlorine substituents. Thus, highly chlorinated compounds such as polychlorinated biphenyls (PCBs), hexachlorobenzene and pentachlorophenol tend to persist in the environment. Biodegradation of highly chlorinated aromatic compounds begins with dechlorination reactions that replace chlorine atoms with hydrogen atoms. Once a sufficient number of chlorine atoms are removed form the ring, these compounds can be degraded relatively quickly. Despite the importance of this type of dechlorination reaction, no mechanistic or structural information is available about the enzymes tht catalyze these reactions. The first part of this proposal describes the first mechanistic experiments of this type of enzyme. A critical question in the field of biodegradation is how microorganisms have evolved the ability to degrade compounds such as pentachlorophenol which were not present in large quantities in the environoment until relatively recently. One approach to this question is to compare the enzymes and corresponding gene sequences in a number of microorganisms. the second part of this proposal focuses on a comparison of dehalogenase enzymes in two different soil microorganisms, Flavobacteium sp. strain ATCC 39723 and Spingomonas sp.strain RA-2. ***