STRUCTURAL STUDIES OF MULTICOMPONENT BACTERIAL MONOOXYGENASES

多组分细菌单加氧酶的结构研究

• 批准号: 8169251
• 负责人: Stephen J. Lippard
• 金额: $ 0.35万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169251
• 关键词: Characteristics; Complement; Complex; Computer Retrieval of Information on Scientific Projects Database; Data; Data Set; Enzymes; Family; Funding; Grant; Hydrocarbons; Institution; Metals; Methane hydroxylase; Mixed Function Oxygenases; Phenol 2-monooxygenase; Protein Binding; Pseudomonas; Publications; Reporting; Research; Research Personnel; Resolution; Resources; Role; Site; Site-Directed Mutagenesis; Source; Structure; United States National Institutes of Health; Work; carboxylate; dimer; divalent metal; genetic regulatory protein; improved; mutant; reconstitution; toluene 2-xylene monooxygenase

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Bacterial multicomponent monooxygenases are a family of enzymes that utilize a carboxylate-bridged diiron center to hydroxyalate a variety of hydrocarbon substrates. Essential for this activity is the formation of a complex between the hydroxylase and regulatory protein. The structure of the Pseudomonas sp. OX1 phenol hydroxylase, PHH-PHM complex was determined to 2.3 ¿ resolution. The regulatory protein binds on helices A, E, and F of the hydroxylase alpha subunit at the dimer interface 12 ¿ above the diiron center. Although the metal center resembled the structure of mixed-valent methane monooxygenase, significant structural changes were observed in helices E and F that were different than the configuration typically observed in the uncomplexed form of the hydroxylase. These changes have implications for substrate activation and substrate selectivity, the significance of which is currently being explored. Since determining this structure, structural work has focused on obtaining higher resolution data on the PHH-PHM complex with improved regulatory protein occupancy, and crystallographic characterization of various mutants of PHH and toluene/o-xylene monooxygenase hydroxylase from Pseudomonas sp. OX1. Higher resolution and structures of the complex with improved regulatory protein occupancy could confirm mechanistic conclusions and speculations about the structural effects of regulatory protein binding proposed upon analysis of the initial PHH-PHM data. Similarly, a structure of the hydroxylase in the absence of the regulatory protein may reveal critical, mechanistically relevant characteristics of the hydroxylase structure while also providing a platform to further investigate the structural effects of regulatory protein binding on the hydroxylase through comparison with the PHH-PHM structure. Diffraction data from these projects are only preliminary and require improvement and analysis before they may be reported. In effort to obtain a diferric or analogous structure of PHH or PHH-PHM, dithionite soaked divalent metal reconstituted and crystals of both species were pursued but not yet successfully obtained such to yield a quality diffraction data set for structure determination. Mechanistic data on mutant forms of ToMOH (T201X) and PHH (N204X) in which conserved residues near the diiron site were varied by site-directed-mutagenesis indicate roles for these residues, so the mutants were crystallized and their diffraction data collected to yield structural information that complements the mechanistic studies. This structural information is presently being analyzed and prepared for publication in conjuction with the relevant mechanistic studies and their results.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 细菌多成分单加氧酶是一种利用羧酸酯桥的二铁中心的酶家族，用于羟基羟基盐酸盐。这种活性的必不可少的是羟化酶和调节蛋白之间的复合物的形成。假单胞菌的结构。 OX1苯酚羟化酶，PHH-PHM复合物确定为2.3»分辨率。调节蛋白在二聚体界面上的羟化酶Alpha亚基的螺旋A，E和F上结合，尽管金属中心与混合价值甲烷单加尝试酶的结构相似，但在螺旋E和F中观察到与典型的构型相同的螺旋e和F中观察到的显着结构变化。这些变化对底物激活和底物选择性具有影响，目前正在探索其意义。自从确定这种结构以来，结构性工作一直集中在获得PHH-PHM复合物上的更高分辨率数据，并改善了调节蛋白占用率，以及PHH和甲苯/O-二甲苯单加氧酶羟化酶羟化酶的各种突变体的晶体表征从pseudomonas sp获得。 OX1。该复合物的更高分辨率和结构具有改进的调节蛋白占用性，可以证实有关在分析初始PHH-PHM数据时提出的调节蛋白结合的结构效应的机理结论和猜测。同样，在没有调节蛋白的情况下，羟化酶的结构可能会揭示羟化酶结构的重要，机械相关的特征，同时还提供了一个平台，以进一步研究调节蛋白结合对羟化酶的结构效应，通过与PHH-PHM结构进行比较。这些项目的衍射数据仅是初步的，需要改进和分析才能报告。为了获得PHH或PHH-PHM的差异或类似结构，追捕了二硫代石的二硫代矿石重构和两种物种的晶体，但尚未成功获得，以产生用于结构确定的质量衍射数据集。关于TOMOH突变形式（T201X）和PHH（N204X）的机械数据，其中构成在Diron位点附近保留的保留的机械数据通过位置定向的 - 氧化作用变化，表明这些残留物的作用，因此将突变体结晶，并收集其衍射数据以产生结构信息，以使结构信息理解机械研究。介绍了这些结构信息，并准备与相关的机械研究及其结果一起出版。