STRUCTURAL STUDIES OF MULTICOMPONENT BACTERIAL MONOOXYGENASES

多组分细菌单加氧酶的结构研究

• 批准号: 7955153
• 负责人: Stephen J. Lippard
• 金额: $ 0.21万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955153
• 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; structural biology; 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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 细菌多组分单加氧酶是一类利用羧酸根桥联的二铁中心对多种烃类底物进行羟醛酸化的酶。这种活性的关键是羟化酶和调节蛋白之间形成复合物。假单胞菌属OX 1苯酚羟化酶PHH-PHM复合物的结构被确定为2.3 ½分辨率。调节蛋白结合在羟化酶α亚基的螺旋A、E和F上，位于二铁中心上方12 ½的二聚体界面处。虽然金属中心类似于混合价甲烷单加氧酶的结构，但在螺旋E和F中观察到显著的结构变化，其不同于在未络合形式的羟化酶中通常观察到的构型。这些变化对底物活化和底物选择性有影响，其意义目前正在探索中。由于确定了这种结构，结构的工作集中在获得更高的分辨率数据的PHH-PHM复合物与改善的调节蛋白质占有率，和晶体学表征的各种突变体的PHH和甲苯/邻二甲苯单加氧酶羟化酶假单胞菌属。更高的分辨率和结构的复合物与改善的调节蛋白的占有率可以确认机制的结论和推测的结构效应的调节蛋白结合后提出的初步PHH-PHM数据分析。类似地，在不存在调节蛋白的情况下羟化酶的结构可以揭示羟化酶结构的关键的、机械相关的特征，同时还提供了通过与PHH-PHM结构进行比较来进一步研究调节蛋白结合对羟化酶的结构影响的平台。来自这些项目的衍射数据只是初步的，在报告之前需要改进和分析。为了获得PHH或PHH-PHM的二铁或类似结构，寻求连二亚硫酸盐浸泡的二价金属重构和两种物质的晶体，但尚未成功获得，从而产生用于结构测定的质量衍射数据集。ToMOH（T201 X）和PHH（N204 X）的突变形式的机制数据，其中通过定点诱变改变二铁位点附近的保守残基，表明这些残基的作用，因此将突变体结晶并收集其衍射数据以产生补充机制研究的结构信息。目前正在分析这些结构信息，并准备与相关的机理研究及其结果一起发表。