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.
