MECHANISMS OF ENZYMATIC OXGEN FIXATION

酶促氧化的机制

• 批准号: 3277586
• 负责人: DAVID T GIBSON
• 金额: $ 18.75万
• 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 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3277586
• 关键词: Escherichia coli; Pseudomonas; X ray crystallography; bacterial genetics; biotransformation; carbopolycyclic compound; electron spin resonance spectroscopy; electron transport; enzyme complex; enzyme mechanism; enzyme structure; genetic recombination; genetic strain; microorganism metabolism; molecular cloning; monoclonal antibody; naphthalenes; nucleic acid sequence; oxidation; oxygenases; protein reconstitution; spectrometry; toluene

The major objective of this proposal is to elucidate the mechanisms used by bacteria to initiate the degradation of aromatic hydrocarbons. These studies will continue to provide, basic health-related information on oxygen fixation, the biodegradation of environmental pollutants and the development of new procedures for the synthesis of pharmaceutical products such as prostaglandins, inositol phosphates and anti tumor compounds. Pseudomonas putida F1 and Pseudomonas sp. NCIB 9816 both utilize multicomponent enzyme systems to incorporate molecular oxygen into toluene and naphthalene respectively. Toluene dioxygenase consists of three components that participate in the transfer of electrons from NADH to the terminal dioxygenase which oxidizes toluene to enantiomerically-pure (+)- cis-(1S,2R)-dihydroxy-3-methylcyclohexa-3,5-diene. Naphthalene dioxygenase utilizes an analogous system to oxidize naphthalene to (+)-cis-(1R,2S)- dihydroxy-1,2-dihydronaphthalene. The properties of the two enzyme systems are different. The project involves the use of recombinant strains of E. coli, to provide significant quantities of individual proteins for studies on: 1. The protein-protein interactions and mechanisms of electron transport from NADH to the terminal dioxygenases. 2. Electron transfer, substrate binding and subunit interactions in the terminal dioxygenase components of toluene and naphthalene dioxygenases. The procedures involved include chemical coupling, spectrophotometric and electron paramagnetic resonance (EPR) studies in the presence and absence of monoclonal antibodies raised against the individual components of both dioxygenases. 3. The oxidation of indan by the cloned naphthalene dioxygenase system. Products will be isolated by high pressure liquid chromatography and the enantiomeric composition of chiral products will be determined by conventional chemical techniques. 4. The biophysical properties of the iron-sulfur clusters and iron in the molecular events leading to the stereospecific incorporation of molecular oxygen into toluene and naphthalene. These will be collaborative studies with scientists who are acknowledged experts in the following techniques: EPR, magnetic circular dichroism, resonance Ramon, Mossbauer, ENDOR and EXAFS spectroscopies. Additional collaborative studies will focus on the crystallization and use of X-ray diffraction technology to determine the structural properties of the individual components of toluene and naphthalene dioxygenases.

该提案的主要目标是阐明所使用的机制 细菌启动芳香烃的降解。 这些 研究将继续提供与健康相关的基本信息 固氧、环境污染物的生物降解和 开发药品合成新程序 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 均利用 将分子氧结合到甲苯中的多组分酶系统 和萘。 甲苯双加氧酶由三部分组成 参与电子从 NADH 转移到 末端双加氧酶，将甲苯氧化成对映体纯的 (+)- 顺式-(1S,2R)-二羟基-3-甲基环己-3,5-二烯。 萘双加氧酶 利用类似的系统将萘氧化成 (+)-cis-(1R,2S)- 二羟基-1,2-二氢萘。 两种酶系统的特性 是不同的。 该项目涉及使用大肠杆菌重组菌株。 大肠杆菌，为研究提供大量的单个蛋白质 on: 1. 蛋白质-蛋白质相互作用和电子机制 从 NADH 到末端双加氧酶的转运。 2.电子转移， 末端双加氧酶中的底物结合和亚基相互作用 甲苯和萘双加氧酶的成分。 程序 涉及化学偶联、分光光度和电子 在存在和不存在的情况下进行顺磁共振（EPR）研究 针对两者的各个成分产生的单克隆抗体 双加氧酶。 3.克隆萘氧化茚满的反应 双加氧酶系统。 产品将被高压液体隔离 色谱法和手性产物的对映体组成将是 通过常规化学技术测定。 4.生物物理学 铁硫簇和铁在分子事件中的性质 导致分子氧立体定向结合到 甲苯和萘。 这些将是与以下机构的合作研究 科学家是以下技术领域公认的专家：EPR、 磁圆二色性、共振 Ramon、Mossbauer、ENDOR 和 EXAFS 光谱检查。 其他合作研究将集中于 结晶并使用X射线衍射技术测定 甲苯各个组分的结构特性 萘双加氧酶。