The major objective of this research proposal is to elucidate the mechanisms used by bacteria to initiate the degradation of aromatic hydrocarbons. The focal point of our studies is naphthalene dioxygenase (NDO), a multi-component enzyme system in Pseudomonas species NCIMB 9816-4 that adds both atoms of oxygen to naphthalene to form homochiral (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. Fundamental studies on NDO provide health-related information on the mechanisms of oxygen activation; the biodegradation of carcinogenic aromatic hydrocarbons and related environmental pollutants; the development of environmentally benign procedures for the production of useful chemicals and the production of chiral intermediates for the synthesis of single enantiomer pharmaceutical products and other biologically active compounds.
The specific aims of the project focus on the recent determination of the crystal structure on the oxygenase component of NDO. These include: X-ray crystallographic studies on a putative indole-peroxy-iron complex and other oxygenase- substrate/analog complexes; site-directed mutagenesis and kinetic studies to identify amino acids that are involved in determining the regiospecificity and enantiospecificity of the products from naphthalene, biphenyl and phenanthrene; identification of amino acids involved in binding the ferredoxin and oxygenase components of NDO and the mechanism of electron transfer between the Rieske [2Fe-2S] centers in each protein; the rational design of the oxygenase active site to accommodate the binding and elimination of nitrite from nitrotoluenes Electron paramagnetic resonance, stopped-flow spectrophotometric and freeze-quench techniques will be used to identify the intermediates involved in oxygen activation and reaction with naphthalene during turnover of the oxygenase. The genes encoding the enantiospecific toluene and naphthalene dihydrodiol dehydrogenases will be used to construct recombinant strains of Escherichia coli that produce novel homochiral synthons.
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