The objectives of this proposal are to investigate microbial pathways of aliphatic alkene and epoxide metabolism and the properties of the enzymes involved in alkene/epoxide transformations. Aliphatic alkenes, aromatics, and epoxides are widely used in industry, and there is considerable concern over the potential mutagenic and carcinogenic properties of these compounds. Aliphatic alkenes and aromatic compounds are excellent substrates for microsomal cytochromes p450 monooxygenases, and are oxygenated across the double bond to the corresponding epoxides. Epoxides formed in this manner are potent alkylating agents that covalently modify proteins and DNA, thereby giving rise to their mutagenic properties. A number of bacteria are able to grow at the expense of aliphatic alkenes and epoxides as their sole carbon and energy sources in spite of the toxicity and reactivity of these compounds. For two such bacteria, Xanthobacter strain Py2 and Nocardia corallina strain B276, aliphatic epoxides formed from alkene oxidation are further metabolized in a novel ring-opening and carboxylation reaction, catalyzed by a new type of multicomponent carboxylase, that produces B-ketoacids as products. For other alkene-oxidizing bacteria, the pathways and enzymes of aliphatic epoxide metabolism remain uncharacterized. This proposal will advance the understanding of microbial alkene and epoxide metabolism through the accomplishment of the following specific aims: Biochemical, mechanistic and spectroscopic characterization of the epoxide carboxylase components, cofactors, and reaction intermediates. The novel four-component epoxide carboxylase enzyme systems identified and purified in the previous funding cycle will be further characterized. Coenzymes X and N, low molecular weight thiol compounds to which epoxyalkanes are conjugated in the first step of the reaction, will be characterized, as will the conjugated intermediates formed during the course of the reactions. Structural Characterization of epoxide carboxylase. The determination of the three-dimensional structures of the four epoxide carboxylase components from Xanthobacter strain Py2 will be pursued. Characterization of the metabolic pathways and enzymes of ethylene and isoprene metabolism. The pathway(s) by which representative ethylene- and isoprene-utilizing bacteria metabolize these alkenes will be investigated. Special attention will be focused on the characterization of the reactions in which the epoxides formed from alkene epoxidation are converted to the next pathway intermediates. The epoxide-converting enzymes will be purified to homogeneity and characterized.
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