MECHANISMS OF ENZYMATIC OXYGEN FIXATION

酶促固氧机制

• 批准号: 2021888
• 负责人: DAVID T GIBSON
• 金额: $ 20.54万
• 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/2021888
• 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.

本研究计划的主要目标是阐明 细菌使用的机制将分子氧原子添加到 甲苯和萘。 这些研究将继续提供基础 健康相关信息：氧固定机制；这 苯的生物降解；甲苯、乙苯和二甲苯 (BTEX)， 是常见的地下水污染物；以及新手性的形成 合成生物活性化合物的中间体， 例如，前列腺素、磷酸肌醇和抗肿瘤化合物。 恶臭假单胞菌 F1 和假单胞菌 sp. NCIB 9816 均使用 多组分酶系统将甲苯和萘氧化成 对映体纯 (+)-顺-(1S,2R)-二羟基-3-甲基环己-3,5-二烯 和(+)-顺式-(1R,2S)-二羟基-1,2-二氢萘。 对这些和其他芳烃顺二烯二醇作为手性合成子的兴趣 新医药产品的生产可见于合成 过去五年中生产了 180 种芳烃顺式二烯二醇。 该项目包括 使用大肠杆菌重组菌株来产生高水平的 还原酶、铁氧还蛋白（Rieske 型）和铁硫加氧酶（α 和 β亚基）甲苯和萘双加氧酶的成分。 尽管两种双加氧酶的结构组织相似，但它们 其底物特异性表现出根本差异。 这是 在反应中尤其明显，其中唯一的差异在于 反应产物是它们的对映体组合物，例如 甲苯和萘生成(1R)-茚满醇和(1S)-茚满醇 分别是双加氧酶。 纯化的蛋白质和加氧酶亚基 组件将用于 1) 研究蛋白质-蛋白质相互作用 参与电子转移至末端加氧酶； 2）研究 从纯化的 α 和 β 中组装加氧酶成分 亚基； 3）研究杂合加氧酶中β亚基的作用；和 4）对铁氧配合物的性质进行合作研究 参与顺式芳烃二烯二醇的对映特异性形成。 这些 将与公认的专家科学家进行合作研究 在以下生物物理技术中：EPR、2D-NMR、磁环 二色性、共振 Ramon、Mossbauer、ENDOR 和 EXAFS 光谱，以及 X射线晶体学。