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. ***
