Molecular mechanisms of alkane hydroxylase (AlkB) reactivity and selectivity

项目摘要 许多维持生命的过程都需要与大气中的氧气发生反应。III类双铁 蛋白质利用氧选择性地氧化脂质，并将OH基团置于关键的分子中， 生物合成途径III类双铁酶在脂质合成的许多方面起着重要作用 和新陈代谢，并与人类健康问题，包括肥胖，糖尿病，注意力缺陷， 紊乱和神经退化。它们在石油的自然生物修复中也至关重要。有一个 缺乏关于这个膜酶家族的机械信息，主要是因为它们 膜相关的性质使它们很难纯化和研究。烷烃单加氧酶 （AlkB）是与脂肪酸去饱和酶一起沿着的III类整合膜双铁蛋白的成员 和脂肪酸羟化酶。AlkB的氨基酸序列表明它在结构上不相似 其他具有类似功能的酶。确定其三维结构是一项壮举， 几十年来科学家们都没有发现。 在前期工作的重要一步，PI Austin和合作研究者Feng已经解决了 AlkB与结合底物的第一种结构，并表明它作为一个优秀的模型系统， 了解III类二铁蛋白的催化机制。这一突破，加上 建立了一种新的快速功能表征方法，并开发了一套 AlkB活性AlkB同系物，为回答这些重要的关键问题铺平了道路 金属酶PI将整合结构，功能，生物化学，计算和 光谱研究，以确定三维结构的二铁活性位点，确定 底物特异性的决定因素，了解AlkB是如何被其伴侣蛋白激活的，并探索如何 存在共价结合的电子转移伴侣，仅在一类革兰氏阳性 细菌，改变了这个酶家族的反应性。 在这样做的过程中，他们将扩大战略的基本知识，以打破和使关键的化学键， 这可能会导致新的合成路线的发展， 分子。他们的工作还将为靶向这一酶家族的努力提供重要的见解， 治疗目的。

Project Summary Reactions with atmospheric oxygen are required for many life-sustaining processes. The class-III diiron proteins use oxygen to selectively oxidize lipids and to put OH groups into molecules in critical biosynthetic pathways. Class-III diiron enzymes play essential roles in many aspects of lipid synthesis and metabolism and are linked to human health problems including obesity, diabetes, attention-deficit disorder, and neurodegeneration. They are also crucial in the natural bioremediation of oil. There is a dearth of mechanistic information about this family of membrane enzymes, primarily because their membrane-associated nature makes them very difficult to purify and study. Alkane monooxygenase (AlkB) is a member of the class-III integral membrane diiron proteins along with fatty acid desaturases and fatty acid hydroxylases. The amino acid sequence of AlkB indicates that it is not structurally similar to other enzymes with similar functions. Determining its three-dimensional structure is a feat that has eluded scientists for decades. In an important step forward in preliminary work, PI Austin and co-Investigator Feng have solved the first structure of AlkB with a bound substrate and shown that it serves as an excellent model system to understand the catalytic mechanism of class-III diiron proteins. This breakthrough, together with the establishment of a novel assay for rapid functional characterization and the development of a suite of AlkB active AlkB homologs, paves the way to answering key questions about these important metalloenzymes. The PIs will integrate structural, functional, biochemical, computational, and spectroscopic studies to determine the three-dimensional structure of the diiron active site, identify determinants of substrate specificity, learn how AlkB is activated by its partner protein, and probe how the presence of a covalently bound electron-transfer partner, found only in a class of gram positive bacteria, changes the reactivity of this enzyme family. In so doing, they will expand the basic knowledge of strategies to break and make key chemical bonds, which may lead to the development of new synthetic routes to make life-saving and life-extending molecules. Their work will also provide critical insights to efforts to target this family of enzymes for therapeutic purposes.