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。三级双铁 蛋白质利用氧气选择性地氧化脂质，并将羟基置于关键的分子中 生物合成途径。III型双铁酶在脂质合成的许多方面起着至关重要的作用 和新陈代谢，与人类健康问题有关，包括肥胖、糖尿病、注意力缺陷 紊乱和神经退行性变。它们在石油的自然生物修复中也是至关重要的。有一个 缺乏关于这一膜酶家族的机制信息，主要是因为它们的 膜结合的性质使它们的纯化和研究变得非常困难。烷基单加氧酶 (AlkB)是与脂肪酸脱饱和酶一起的III类完整膜双铁蛋白的成员 和脂肪酸羟基酶。AlkB的氨基酸序列表明它在结构上不相似 对其他具有类似功能的酶。确定它的三维结构是一项具有 几十年来，科学家们一直在躲避。 在前期工作向前迈出的重要一步中，皮奥斯汀和合作调查员冯解决了 具有结合底物的AlkB的第一个结构，并表明它是一个很好的模型体系 了解III型双铁蛋白的催化机制。这一突破，连同 一种新的快速功能鉴定方法的建立和一套新的检测方法的建立 AlkB活性AlkB同系物，为回答有关这些重要问题的关键问题铺平了道路 金属酶。PI将把结构、功能、生化、计算和 光谱研究确定二铁活性中心的三维结构，鉴定 底物特异性的决定因素，了解AlkB是如何被其伙伴蛋白激活的，并探索如何 共价结合的电子转移伙伴的存在，只在一类革兰氏阳性中发现 细菌，改变这个酶家族的反应性。 在这样做的过程中，他们将扩展打破和建立关键化学键的策略的基本知识， 这可能会导致开发新的合成路线来拯救生命和延长生命 分子。他们的工作也将为以这种酶家族为目标的努力提供关键的见解 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.