MICROBIAL METABOLISM OF ALIPHATIC ALKENES AND EPOXIDES

这项提案的目的是研究生理学， 微生物代谢的生物化学和遗传学 烯烃、环氧化物和相关的卤代化合物。 有 对生物反应性、毒性和 烯烃和环氧化物的致癌性：环氧化物具有强的烷基化作用 共价修饰蛋白质和DNA的试剂，以及广泛的 烯烃容易被单加氧酶活性活化成环氧化物， 细胞色素P-450酶 许多细菌能够在细菌中生长 脂肪族烯烃和环氧化物作为它们唯一的碳和能量的消耗 来源，包括黄原胶菌株Py 2，一种含有 可诱导乙烯-和丙烯-氧化系统，其将用于 本提案中所述的研究。 在这种细菌中， 烯烃代谢的关键是烯烃单加氧酶， 将烯烃环氧化成其相应的环氧化物，和环氧酶， 其进一步以尚未表征的方式消耗这些环氧化物， 反应 本提案的具体目的是（1）开发体外测定， 纯化至均一，并对烯烃单加氧酶进行生化表征 和环氧酶;（2）研究表达的机制 抑制烯烃单加氧酶、环氧酶和辅助蛋白 （3）测定烯烃和环氧化物的直接产物 代谢，并研究其进一步代谢的途径 作为碳源;（4）分离、克隆和表征编码 烯烃单加氧酶和环氧酶;和（5）进一步表征 烯烃单加氧酶和环氧酶的底物范围，特别是 重点关注脂肪族和卤代烯烃和环氧化物， 它们的毒性和在环境中的出现。 了解细菌能够代谢的机制 烯烃和环氧化物将引起极大的兴趣，因为它们具有诱变性和 这些化合物的致癌潜力。 圆满完成 上述目标将大大有助于我们 了解细菌对异生物质化合物的代谢。

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.