MICROBIAL METABOLISM OF ALIPHATIC ALKENES AND EPOXIDES

这项建议的目的是研究生理学， 脂肪族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。的确有 对生物反应性、毒性和 烯烃和环氧化物的致癌性：环氧化物具有很强的烷基化作用 共价修饰蛋白质和DNA的试剂，以及广泛的 烯烃很容易被单加氧酶活性激活为环氧化物。 细胞色素P-450酶。许多细菌能够在 作为其唯一碳和能量的脂肪族烯烃和环氧化物的费用 来源，包括黄杆菌菌株PY2，一种含有 诱导式乙烯和丙烯氧化系统将用于 这项提案中所描述的研究。在这种细菌中，关键酶 烯烃代谢的关键是烯烃单加氧酶，它催化 烯烃环氧化成其相应的环氧化物，以及环氧酶， 它进一步消耗了这些环氧化物，形成了一种尚不确定的 反应。 这项建议的具体目标是(1)开发体外检测方法， 烯烃单加氧酶的纯化及生化性质研究 和环氧合酶；(2)研究表达的机制(S) 以及对烯烃单加氧酶、环氧合酶和辅助蛋白的抑制 规定；(3)测定烯烃和环氧化物的即刻产物 代谢并研究它们进一步代谢的途径(S) 作为碳源；(4)分离、克隆和鉴定编码基因 烯烃单加氧酶和环氧合酶；以及(5)进一步表征 烯烃单加氧酶和环氧合酶的底物范围，特别是 应重点关注的脂肪族和卤代烯烃及环氧化物 它们的毒性和在环境中的存在。 了解细菌代谢的机制 烯烃和环氧化物将引起人们的极大兴趣，因为 这些化合物的致癌潜力。圆满完成 上述目标将对我们的 了解外源化合物的细菌代谢。

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.