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，Pseudomonas putida（以前是假单胞菌oleovorans gpol）和相关酶二甲氧酶（Xylemiend）的二甲基单加氧酶（Xylme）（Xylme）中的二甲基二氧酶（Xylme）均具有良好的di，在许多细菌中发现了AIKB，包括许多病原体C属。结核分枝杆菌，肺炎军团菌和铜绿假单胞菌的基因均具有与AIKB同源性高的蛋白质相关的基因。该区域授予探讨了活跃位点结构会影响二铁羟基氧化烷烃的机制的假设。具体而言，它检验了以下假设：在AIKB和Xylm的活性位点发现的较软的氮供体的存在会改变这些酶的活性中间体相对于SMMO和T4MOH活性中间体的反应性的反应性，而Smmo和T4MOH的反应性含有含氧型含量富含氧气的辅助环境。它还检验了以下假设：活性位点中的疏水底物分子可能会改变AIKB的反应性。这些假设通过稳定活跃的纯化膜纺丝二烷基非血红素酶来检验，使用一套诊断底物来表征其羟基化机制，并使用同一诊断物的同一套件的非头部生物元素模型的羟基生物元素模型的机制与其机制进行比较。在项目中，对博士后同学和本科研究人员的培训在项目中的旨在众所周知。