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

该项目的具体目的是研究用于降解两种卤代芳烃的生化途径，即2,4,5-三氯苯酚的衍生物除草剂2,4,5-三氯酚氧乙酸酯（2,4,5- t）和杀菌剂五氯苯酚（PCP）。PI和合作者已经鉴定、纯化和表征了洋葱伯克霍尔德菌（假单胞菌）AC 1100和鞘单胞菌（黄杆菌）sp.菌株ATCC 39723降解2,4,5- t和PCP的三种酶，但我们对2,4,5- t和PCP降解途径的了解仍然存在空白。在切割环之前，2,4,5- t和PCP都先完全脱卤并转化为羟基喹啉（1,2,4-三羟基苯），但这两种体系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，然后由氯酚4-单加氧酶氧化为2,5-二氯对对苯二酚，再氧化为5-氯羟基喹啉（5-氯-1,2,4-三羟基苯）。一个氯和一个氢被一种新发现的酶从5-氯羟基喹啉中除去，形成羟基苯醌，羟基苯醌可能被一种尚未发现的醌还原酶还原为羟基喹啉。pcp4 -单加氧酶将PCP氧化为四氯-对对苯二酚，然后四氯-对对苯二酚脱卤酶将四氯-对对苯二酚还原为2,3,6-三氯-对对苯二酚，然后是2,6-二氯-对对苯二酚。2,6-二氯对对苯二酚氯水解酶将2,6-二氯对对苯二酚转化为6-氯羟基喹啉（6-氯-1,2,4-三羟基苯）。目前尚不清楚6-氯羟基喹啉是如何合成羟基喹啉的，但部分表征的双加氧酶只氧化羟基喹啉。本研究将重点关注这些途径中未记录的步骤。他们将鉴定、纯化和表征催化2,4,5- t或PCP降解中未定义反应的酶，就像他们对这两种系统的其他酶所做的那样。纯化蛋白质后，将确定部分氨基酸序列（可能来自n端）。该序列将用于克隆编码每种蛋白质的基因，要么通过将其识别为在另一环境中已知的基因，要么使用基于氨基酸序列的探针。这将使我们能够更完整地描述每种蛋白质的特征，以便了解可能限制其活性的因素。卤化芳香族化合物被广泛用作杀虫剂和除草剂，以及作为木浆漂白等过程的副产品而生产，已大量释放到环境中。它们是主要的环境污染物。它们通常在自然界中没有对应物，而且往往非常稳定。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清除。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有特征。这项研究将发现新的酶，并确定微生物如何利用这些酶来降解卤化化合物。这项工作在生物修复中具有重要的应用价值。

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.