MECHANISMS OF ENZYMATIC OXYGEN FIXATION

酶促固氧机制

• 批准号: 2608787
• 负责人: DAVID T GIBSON
• 金额: $ 21.13万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-08-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2608787
• 关键词: Escherichia coli; Pseudomonas; Raman spectrometry; X ray crystallography; bacterial genetics; biotransformation; circular dichroism; electron spin resonance spectroscopy; electron transport; enzyme complex; enzyme mechanism; enzyme structure; ferredoxin; intermolecular interaction; microorganism metabolism; naphthalenes; nuclear magnetic resonance spectroscopy; nucleic acid sequence; oxidation; oxygenases; toluene

The major objective of this research proposal is to elucidate the mechanisms used by bacteria to add both atoms of molecular oxygen to toluene and naphthalene. These studies will continue to provide basic health related information on: the mechanisms of oxygen fixation; the biodegradation of benzene; toluene, ethylbenzene and xylenes (BTEX), which are common ground water pollutants; and the formation of new chiral intermediates for the synthesis of biologically-active compounds, for example, prostaglandins, inositol phosphates and anti-tumor compounds. Pseudomonas putida F1 and Pseudomonas sp. NCIB 9816 both use multicomponent enzyme systems to oxidize toluene and naphthalene to enantiomerically-pure (+)-cis-(1S,2R)-dihydroxy-3-methylcyclohexa-3,5-diene and (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene respectively. Interest in these and other arene cis-diene diols as chiral synthons for the production of new pharmaceutical products can be seen in the synthesis of 180 arene cis-diene diols in the past five years. The project includes the use of recombinant strains of E. coli to produce high levels of the reductase, ferredoxin (Rieske-type) and iron-sulfur oxygenase (alpha and beta subunits) components of toluene and naphthalene dioxygenases. Although the structural organization of both dioxygenases are similar they show fundamental differences in their substrate specificities. This is particularly evident in reactions where the only differences in the reaction products are their enantiomeric compositions, for example, the formation of (1R)-indanol and (1S)-indanol by toluene and naphthalene dioxygenase respectively. Purified proteins and subunits of the oxygenase components will be used to 1) study the protein-protein interactions involved in electron transfer to the terminal oxygenases; 2) study the assembly of the oxygenase components from their purified alpha and beta subunits; 3) study the role of the beta subunits in hybrid oxygenases; and 4) conduct collaborative studies on the nature of the iron-oxygen complex involved in the enantiospecific formation of cis-arene-diene diols. These collaborative studies will be with scientists who are acknowledged experts in the following biophysical techniques: EPR, 2D-NMR, magnetic circular dichroism, resonance Ramon, Mossbauer, ENDOR and EXAFS spectroscopies, and X-ray crystallography.

该研究建议的主要目的是阐明 细菌用来添加两个分子氧原子的机制 甲苯和萘。 这些研究将继续提供基本 健康相关的信息：氧气固定机制；这 苯的生物降解；甲苯，乙烯苯和二甲苯（BTEX），它们 是常见的地下水污染物；和新手性的形成 用于合成生物活性化合物的中间体，用于 例如前列腺素，肌醇磷酸盐和抗肿瘤化合物。 假单胞菌Putida F1和Pseudomonas sp。 NCIB 9816都使用 多组分酶系统氧化甲苯和萘至 对映体纯化（+） - 顺式 - （1s，2r）-dihydroxy-3-甲基环甲基糖exa-3,5-二烯 和（+） - 顺式 - （1r，2s）-dihydroxy-1,2-二脑映射。 对这些和其他Arene CIS-二烯二醇作为手性合成子的兴趣 在合成中可以看到新药的生产 在过去五年中，有180种Arene Cis-Diene二醇。 该项目包括 大肠杆菌的重组菌株的使用来产生高水平的 还原酶，铁氧还蛋白（Rieske型）和铁硫氧酶（α和 β亚基）甲苯和萘二氧酶的成分。 尽管两加氧酶的结构组织都相似 显示其底物特异性的根本差异。 这是 在反应中尤其明显 反应产物是它们的对映体组成，例如 甲苯和萘的（1R） - indanol和（1s） - indanol的形成 二氧酶分别。 纯化的蛋白质和氧合酶的亚基 组件将用于1）研究蛋白质 - 蛋白质相互作用 参与电子转移到末端氧酶； 2）研究 从其纯化的α和β的氧合酶组成的组装 亚基； 3）研究β亚基在杂化加氧酶中的作用；和 4）进行有关铁氧复合物质性质的合作研究 参与顺式 - 芳烃二醇的对映体形成。 这些 合作研究将与受到认可专家的科学家进行 在以下生物物理技术中：EPR，2D-NMR，磁圆形 二分性能，共鸣拉蒙，莫斯鲍尔，Endor和Exafs Spectroscopies，以及 X射线晶体学。