Characterizing hydroxylation mechanism of diiron enzymes

描述(由申请人提供)：AIkB，恶臭假单胞菌(前身为油假单胞菌GPol)的完整膜二铁烷烃单加氧酶和相关酶二甲苯单加氧酶(使二甲苯的甲基羟化)(XylM)是特征最差的二铁羟基酶。AIkB存在于多种细菌中，包括许多病原菌C属。结核分枝杆菌、嗜肺军团菌和铜绿假单胞菌都有编码与AIkB高度同源的蛋白质的基因。这一领域的拨款探索了活性中心结构影响双铁羟基酶氧化烷烃的机制的假说。具体地说，它测试了这样的假设，即在AIkB和XylM的活性部位发现的较软的氮供体的存在改变了这些酶的活性中间体的反应活性，相对于sMMO和T4MOH的活性中间体的反应，这两个中间体包含了它们的双铁中心的富氧配位环境。它还验证了活性部位的疏水底物分子可能改变AIkB的反应性的假设。通过稳定活性的跨膜二铁非血红素酶，使用一套诊断底物表征它们的羟化机制，并使用同一套诊断底物将它们的机制与非血红素双铁仿生模型的羟化机制进行比较，验证了这些假说。对博士后助理和本科生研究人员的培训在这些项目的目标中占有突出地位。

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.