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: 6417525
• 负责人: Scott A. ENSIGN
• 金额: $ 6.33万
• 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 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6417525
• 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.

该提案的目的是研究微生物途径， 脂肪族烯烃和环氧化物代谢及酶的性质 参与烯烃/环氧化物转化。脂肪族烯烃、芳香族化合物和 环氧化物在工业中被广泛使用， 这些化合物的潜在致突变性和致癌性。 脂肪族烯烃和芳香族化合物是用于聚合的优良底物。 微粒体细胞色素p450单加氧酶，并通过 与相应的环氧化物形成双键。以这种方式形成的环氧化物是 共价修饰蛋白质和DNA的有效烷化剂， 从而产生了它们的诱变特性。许多细菌能够 以牺牲脂肪族烯烃和环氧化物作为其唯一碳而生长， 能源，尽管这些化合物的毒性和反应性。为 两种这样的细菌，黄曲霉菌株Py 2和诺卡氏菌菌株B276， 由烯烃氧化形成的脂族环氧化物进一步代谢为 新型的开环和羧化反应，由一种新型的催化剂催化， 多组分羧化酶，其产生β-酮酸作为产物。为其他 烯烃氧化细菌，脂肪环氧化途径和酶 代谢仍然没有特征。这一建议将促进理解 微生物烯烃和环氧化物代谢的完成， 具体目标如下： 环氧化物的生化、机理和光谱表征 羧化酶组分、辅因子和反应中间体。小说 四组分环氧化物羧化酶酶系统鉴定和纯化， 上一个供资周期的特点将得到进一步说明。辅酶X和N， 低分子量硫醇化合物，环氧烷烃与其共轭， 反应的第一步，将被表征，如将共轭 在反应过程中形成的中间体。 环氧羧化酶的结构表征。的确定 四种环氧化物羧化酶组分的三维结构 将追踪黄曲霉菌株Py 2。 乙烯和异戊二烯的代谢途径和酶的表征 新陈代谢.代表性的乙烯-和乙烯-二聚体通过的途径是： 将研究代谢这些烯烃异戊二烯利用细菌。 特别注意将集中在表征的反应， 其中由烯烃环氧化形成的环氧化物被转化为下一个 途径中间体。将纯化环氧化物转化酶， 同质性和特征。