MICROBIAL METABOLISM OF ALIPHATIC ALKENES AND EPOXIDES

脂肪族烯烃和环氧化物的微生物代谢

• 批准号: 6951327
• 负责人: Scott A. ENSIGN
• 金额: $ 6.26万
• 依托单位: UTAH STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6951327
• 关键词: Actinomycetales; Mycobacterium; alkenes; computer assisted sequence analysis; electron spin resonance spectroscopy; enzyme induction /repression; enzyme mechanism; epoxides; gene induction /repression; genetic regulation; gram negative bacteria; halocarbon compound; microorganism genetics; microorganism metabolism; molecular cloning; nucleic acid sequence; protein purification; ultraviolet spectrometry

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.

该提案的目标是研究 脂肪烷烯烃和环氧代谢以及酶的特性 参与烯烃/环氧化物的转化。脂肪烷烷烃，芳香剂和 环氧化物在行业中广泛使用，对 这些化合物的潜在诱变和致癌特性。 脂肪族烷烃和芳香化合物是极好的底物 微粒体细胞色素P450单加氧酶，并在整个 与相应的环氧化物双键。以这种方式形成的环氧化物是 有效的烷基化剂，可将蛋白质和DNA共价，从而 产生其诱变特性。许多细菌能够 以脂肪族烷烃和环氧化物为代价生长为唯一的碳和 尽管这些化合物具有毒性和反应性，但能源源。为了 两个这样的细菌，Xanthobacter菌株Py2和Nocardia corallina菌株B276， 由烯烃氧化形成的脂肪族环氧化物在A中进一步代谢 新型环和羧化反应，由一种新型的新型 多组分羧化酶，产生B-酮酸作为产品。其他 烯烃氧化细菌，脂肪族环氧的途径和酶 代谢仍然没有特征。该建议将提高理解 通过完成的微生物烯烃和环氧代谢 遵循特定目的： 环氧的生化，机械和光谱表征 羧化酶成分，辅助因子和反应中间体。小说 在鉴定和纯化的四成分环氧化羧化酶系统中 先前的资金周期将进一步表征。辅酶X和N， 低分子量的硫醇化合物，环氧基因缀合的硫醇化合物 反应的第一步将被描述，共轭也将被表征 反应过程中形成的中间体。 环氧羧化酶的结构表征。确定 来自四个环氧羧化酶成分的三维结构 将追求Xanthobacter菌株Py2。 乙烯和异戊二烯的代谢途径和酶的表征 代谢。代表性乙烯和 将研究这些烷烃的等法利用细菌代谢。 特别关注将集中在反应的表征上 由烯烃环氧化形成的环氧化物转化为下一个 途径中间体。环氧转化酶将被纯化为 同质性和特征。