One of the major health problems facing the world today is the massive amount of toxic waste present in our environment. Several strategies are being developed to eliminate these from the biosphere. A promising method is bioremediation. Biodegradation of polyaromatic compounds, polychlorinated biphenyls etc. is carried out by several strains of bacteria. The first step in the degradation of these compounds is dihydroxylation of the aromatic ring. This reaction is catalyzed by the multi-component Rieske non-heme iron oxygenases, which are the focus of this proposal. These enzymes add both atoms of molecular oxygen to aromatic hydrocarbons in a stereo- and regio-specific fashion. This feature is and can be used in the production of chiral synthons, which then form excellent platforms for enantiopure drug production. They also catalyze a wide variety of reactions on a broad range of substrates. These enzymes can be used in the environmentally benign synthesis of several hard to synthesize compounds, contributing to green chemistry. The ability to manipulate and engineer these enzymes is hence of great environmental, medical and commercial importance. However doing this in a rational fashion depends on our understanding of the structural basis of their function. There is a critical lack of detailed structural information among this class of dioxygenases. The only dioxygenase structure known is that of naphthalene dioxygenase from Pseudomonas sp. NCIB 981 6-4. The goal of this proposal is to provide a structural framework that can be used to interpret existing functional results as well as provide a basis to design further functional studies. The principal investigator plans to 1) determine the structure of 2-nitrotoluene dioxygenase from Pseudomonas sp. strain JS42 and naphthalene dioxygenase from Rhodococcus sp. strain NCIMB 12038, the reductase and ferredoxin components of the naphthalene dioxygenase and 2-nitrotoluene dioxygenase systems in Pseudomonas; 2) elucidate by determining and analyzing the structure of substrate and product complexes of wild type and mutant Pseudomonas sp naphthalene dioxygenase the structural basis for modified regio-specificity of product formation; 3) map the regions of protein-protein interactions between the reductase, ferredoxin, and dioxygenase components by a combination of mutagenesis, structural and solution studies. These results should provide a better insight into the biochemistry of Rieske non-heme iron oxygenases.
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