Characterizing hydroxylation mechanism of diiron enzymes

表征二铁酶的羟基化机制

• 批准号: 6847374
• 负责人: RACHEL Narehood AUSTIN
• 金额: $ 20.87万
• 依托单位: BATES COLLEGE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6847374
• 关键词: Pseudomonas; active sites; alkanes; bacterial proteins; biomimetics; enzyme activity; enzyme mechanism; enzyme structure; high performance liquid chromatography; hydroxylation; iron compounds; organometallic compounds; oxygenases; protein purification; rubredoxins; spectrometry

DESCRIPTION (provided by applicant): AIkB, the integral membrane diiron alkane monooxgenase from Pseudomonas putida (formerly Pseudomonas oleovorans GPol) and the related enzyme xylene monooxygenase (which hydroxylates the methyl group of xylene) (XylM) are among the least well characterized diiron hydroxylases. AIkB is found in numerous bacteria, including many pathogen c genera. Mycobacterium tuberculosis, Legionella pneumophilia, and Pseudomonas aeruginosa all have genes that code for proteins with high homology to AIkB. This AREA grant explores the hypothesis that active site structure affects the mechanisms by which diiron hydroxylases oxidize alkanes. Specifically, it tests the hypothesis that the presence of the softer nitrogen donors found in the active sites of AIkB and XylM alters the reactivity of the active intermediate of these enzymes relative to the reactivity of the active intermediate of sMMO and T4MOH, which contain oxygen rich coordination environments for their diiron centers. It also tests the hypothesis that hydrophobic substrate molecules in the active site may alter the reactivity of AIkB. These hypotheses are tested by stabilizing active purified membrane-spanning diiron non-heme enzymes, characterizing their hydroxylation mechanisms using a suite of diagnostic substrates and comparing their mechanisms to the mechanism of hydroxylation of a non-heme diiron biomimetic model, using the same suite of diagnostic substrates. The training of a post-doctoral associate and undergraduate researchers figures prominently in the projects aims.

描述（由申请人提供）：AIkB，来自恶臭假单胞菌（以前的食油假单胞菌GPol）的整合膜二铁烷烃单加氧酶和相关酶二甲苯单加氧酶（其羟基化二甲苯的甲基）（XylM）是最不充分表征的二铁羟化酶。AIkB存在于许多细菌中，包括许多病原体属。结核分枝杆菌、嗜肺军团菌和铜绿假单胞菌都具有编码与AIkB具有高度同源性的蛋白质的基因。这个区域补助金探索的假设，活性部位结构影响的机制，二铁羟化酶氧化烷烃。具体地，它测试了这样的假设，即在AIkB和XylM的活性位点中发现的较软氮供体的存在改变了这些酶的活性中间体相对于sMMO和T4MOH的活性中间体的反应性的反应性，sMMO和T4MOH的活性中间体含有用于其二铁中心的富氧配位环境。它还测试了活性位点中的疏水底物分子可能改变AIkB的反应性的假设。这些假设进行了测试，通过稳定活性纯化的跨膜二铁非血红素酶，其羟基化机制的特征，使用一套诊断底物和比较其机制的非血红素二铁仿生模型的羟基化机制，使用同一套诊断底物。博士后助理和本科生研究人员的培训在项目目标中占有突出地位。