The objective of this proposal is to investigate the physiology, biochemistry, and genetics of the microbial metabolism of aliphatic alkenes, epoxides and related halogenated compounds. There is considerable concern about the biological reactivity, toxicity and carcinogenicity of alkenes and epoxides: epoxides are potent alkylating agents which covalently modify proteins and DNA, and a broad range of alkenes are readily activated to epoxides by the monooxygenase activity of cytochrome P-450 enzymes. A number of bacteria are able to grow at the expense of aliphatic alkenes and epoxides as their sole carbon and energy source, including Xanthobacter strain Py2, a soil bacterium containing an inducible ethylene- and propylene-oxidizing system which will be used for the studies described in this proposal. In this bacterium the key enzymes of alkene metabolism are alkene monooxygenase, which catalyzes the epoxidation of alkenes to their corresponding epoxides, and an epoxidase, which further consumes these epoxides in an as yet uncharacterized reaction.
The specific aims of this proposal are to (1) develop in vitro assays for, purify to homogeneity, and biochemically characterize alkene monooxygenase and epoxidase; (2) investigate the mechanism(s) by which the expression and repression of alkene monooxygenase, epoxidase, and accessory proteins is regulated; (3) determine the immediate products of alkene and epoxide metabolism and study the pathway(s) by which they are further metabolized as carbon sources; (4) isolate, clone, and characterize the genes encoding alkene monooxygenase and epoxidase; and (5) further characterize the substrate ranges of alkene monooxygenase and epoxidase, with particular emphasis on aliphatic and halogenated alkenes and epoxides of concern due to their toxicity and occurrence in the environment. Understanding the mechanisms by which bacteria are able to metabolize alkenes and epoxides will be of great interest, given the mutagenic and carcinogenic potential of these compounds. The successful completion of the objectives outlined above will contribute significantly to our understanding of bacterial metabolism of xenobiotic compounds.
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