Biochemistry of 2,4,5-trichlorophenoxyacetate and pentachlorophenol degradation

9722970 Luying Xun该项目的具体目标是研究用于降解两种卤代芳烃的生物化学途径，即除草剂2，4，5-三氯苯氧基乙酸酯（2，4，5-T），2，4，5-三氯苯酚的衍生物和生物杀灭剂五氯苯酚（PCP）。PI及其合作者已经鉴定、纯化并表征了洋葱伯克霍尔德氏菌（假单胞菌）菌株AC 1100降解2，4，5-T所涉及的三种酶和鞘氨醇单胞菌（黄杆菌）菌株ATCC 39723降解PCP所涉及的四种酶，但我们对2，4，5-T和PCP降解途径的了解仍存在空白。2，4，5-T和PCP在开环之前都首先完全脱卤并转化为羟基喹啉（1，2，4-三羟基苯），但两个系统中涉及的脱卤酶非常不同。2，4，5-T加氧酶将2，4，5-T氧化为2，4，5-三氯苯酚，2，4，5-三氯苯酚被氯苯酚4-单加氧酶氧化为2，5-二氯对苯二酚，然后氧化为5-氯羟基喹啉（5-氯-1，2，4-三羟基苯）。通过新鉴定的酶从5-氯羟基喹啉中除去氯和氢以形成羟基苯醌，羟基苯醌可能通过尚未鉴定的醌还原酶还原为羟基喹啉。PCP 4-单加氧酶将PCP氧化为四氯对苯二酚，然后四氯对苯二酚脱卤酶将四氯对苯二酚还原脱卤，首先脱卤为2，3，6-三氯对苯二酚，然后脱卤为2，6-二氯对苯二酚。2，6-二氯对苯二酚氯水解酶将2，6-二氯对苯二酚转化为6-氯羟基喹啉（6-氯-1，2，4-三羟基苯）。目前尚不清楚6-氯羟基喹啉是如何被转化为羟基喹啉的，但部分表征的双加氧酶仅氧化羟基喹啉。这项研究将集中在这些途径中没有记录的步骤。他们将鉴定、纯化和表征催化参与2，4，5-T或PCP降解的未定义反应的酶，因为它们与这两个系统的其他酶一样。蛋白质纯化后，将测定部分氨基酸序列（可能来自N-末端）。该序列将用于克隆编码每种蛋白质的基因，或者通过将其鉴定为另一种背景下已知的基因，或者通过使用基于氨基酸序列的探针。这将使我们能够更完整地表征每种蛋白质，以了解可能限制其活性的因素。由于卤代芳族化合物作为杀虫剂和除草剂的广泛使用以及它们作为诸如木浆漂白的工艺的副产物的产生，卤代芳族化合物已经被大量释放到环境中。它们是环境污染物中的一大类。它们通常在自然界中没有对应物，并且通常非常稳定。幸运的是，一些微生物可以降解这些化合物并将其从环境中去除。催化逐步反应以去除卤素（氯、氟和溴）以产生天然代谢中间体的酶很少被表征。这项研究将发现新的酶，并确定微生物如何利用这些酶降解卤代化合物。这项工作可能在生物修复中有重要的应用。

9722970 Luying Xun The specific aims of this project are to study the biochemical pathways used to degrade two halogenated aromatic hydrocarbons, the herbicide 2,4,5-trichlorophenoxyacetate (2,4,5-T), a derivative of 2,4,5-trichlorophenol, and the biocide pentachlorophenol (PCP). The PI and collaborators have identified, purified, and characterized three enzymes involved in 2,4,5-T degradation by Burkholderia (Pseudormonas) cepacia strain AC 1100 and four enzymes involved in PCP degradation by Sphingomonas (Flavobacterium) sp.strain ATCC 39723, but this still leaves gaps in our knowledge of the pathways of both 2,4,5-T and PCP degradation. Both 2,4,5-T and PCP are first completely dehalogenated and converted to hydroxyquinol (1,2,4-trihydroxybenzene) before ring-cleavage, but the dehalogenases involved in the two systems are very different. 2,4,5-T oxygenase oxidizes 2,4,5-T to 2,4,5-trichlorophenol, which is oxidized by chlorophenol 4-monooxygenase to 2,5- dichloro-p-hydroquinone and then to 5-chlorohydroxyquinol (5-chloro-1,2,4-trihydroxybenzene). A chlorine and a hydrogen are eliminated from 5-chlorohydroxyquinol by a newly identified enzyme to form hydroxybenzoquinone which is likely reduced to hydroxyquinol by a yet to be identified quinone reductase. PCP 4-monooxygenase oxidizes PCP to tetrachloro-p-hydroquinone, then tetrachloro-p-hydroquinone dehalogenase reductively dehalogenates tetrachloro-p-hydroquinone, first to 2,3,6-trichloro-p-hydroquinone and then to 2,6-dichloro-p-hydroquinone. 2,6-Dichloro-p-hydroquinone chlorohydrolase converts 2,6-dichloro-p-hydroquinone to 6-chlorohydroxyquinol (6-chloro-1,2,4-trihydroxybenzene). It is unclear how 6-chlorohydroxyquinol is convened to hydroxyquinol, but a partially characterized dioxygenase oxidized only hydroxyquinol. This research will focus on the undocumented steps in these pathways. They will identify, purify and characterize the enzymes that catalyze undefined reactions involved in either 2,4,5-T or PCP degradation, as they have don e with other enzymes of these two systems. After a protein is purified, a partial amino acid sequence (probably from the N-terminus) will be determined. This sequence will be used to clone the gene encoding each protein, either by identifying it as a gene known in another context or by using probes based on the amino acid sequence. This should allow us to more completely characterize each protein in order to understand the factors that might limit its activity. Halogenated aromatic compounds have been released into the environment in large quantities as a result of their wide use as pesticides and herbicides and their production as byproducts of processes such as wood pulp bleaching. They are a major group of environmental pollutants. They usually do not have counterparts in nature and often are very stable. Fortunately, some microorganisms can degrade these compounds and remove them from the environment. Few of the enzymes that catalyze step-by-step reactions to remove the halogens (chlorine, fluorine and bromine) to produce natural metabolic intermediates have been characterized. This research will discover novel enzymes and determine how microorganisms use these enzymes to degrade halogenated compounds. This work may have important applications in bioremediation.