Characterizing hydroxylation mechanism of diiron enzymes

描述（由申请人提供）：AIkB，来自恶臭假单胞菌（以前的伪单胞菌GPol）的整体膜双铁烷烃单加氧酶和相关的二甲苯单加氧酶（羟基化二甲苯的甲基）（XylM）是最不清楚表征的二铁羟化酶之一。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有编码与AIkB高度同源的蛋白的基因。本区域资助探讨了活性位点结构影响二铁羟化酶氧化烷烃机制的假设。具体来说，它验证了AIkB和XylM活性位点中较软的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假设，sMMO和T4MOH的活性中间体具有富氧的配位环境。它也验证了活性位点疏水底物分子可能改变AIkB反应性的假设。这些假设是通过稳定活性纯化跨膜双铁非血红素酶来验证的，使用一套诊断底物来表征它们的羟基化机制，并将它们的机制与使用同一套诊断底物的非血红素双铁仿生模型的羟基化机制进行比较。培养博士后和本科研究人员在项目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.