Molecular mechanisms of alkane hydroxylase (AlkB) reactivity and selectivity

烷烃羟化酶 (AlkB) 反应性和选择性的分子机制

基本信息

批准号：
10451683
负责人：
RACHEL Narehood AUSTIN
金额：
$ 29.17万
依托单位：
BARNARD COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-15 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10451683
关键词：
Active Sites Affect Alcohols Alkane 1-monooxygenase Alkanes Amino Acid Sequence Architecture Attention Deficit Disorder Bacteria Behavior Biochemical Biological Biological Assay Biological Models Bioremediations Biotechnology Carboxylic Acids Chemistry Chimeric Proteins Comparative Study Complex Computer Simulation Crystallization Development Diabetes Mellitus Electron Transport Elements Engineering Environment Enzyme Reactivation Enzymes Escherichia coli Eukaryotic Cell Family Fatty Acid Desaturases Fatty Acids Future Gram-Positive Bacteria Health Human Hydrogen Bonding Iron Knowledge Lead Learning Length Life Light Link Lipids Malignant Neoplasms Maps Mediating Membrane Membrane Proteins Metabolism Metals Mixed Function Oxygenases Modeling Molecular Nature Nerve Degeneration Nitrogen Obesity Oils Oxidation-Reduction Oxygen Oxygenases Pathogenicity Pathway interactions Physiological Play Positioning Attribute Process Protein Family Proteins Reaction Red Algae Research Personnel Resolution Role Route Rubredoxins Savings Scientist Structure Substrate Specificity Testing Therapeutic Work Zinc austin biophysical analysis chemical bond clinically relevant computer studies experimental study insight member metalloenzyme mutant novel overexpression oxidized lipid protein folding rapid test spectroscopic survey therapeutic enzyme therapeutic target three dimensional structure

项目摘要

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.

项目概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级二铁蛋白质利用氧气选择性地氧化脂质，并将 OH 基团放入关键的分子中生物合成途径。 III 类二铁酶在脂质合成的许多方面发挥着重要作用和新陈代谢，并与肥胖、糖尿病、注意力缺陷等人类健康问题有关紊乱和神经退行性疾病。它们对于石油的自然生物修复也至关重要。有一个缺乏关于该膜酶家族的机制信息，主要是因为它们与膜相关的性质使得它们非常难以纯化和研究。烷烃单加氧酶 (AlkB) 是 III 类整合膜二铁蛋白以及脂肪酸去饱和酶的成员和脂肪酸羟化酶。 AlkB的氨基酸序列表明其结构并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一直困扰着科学家。在前期工作中向前迈出了重要的一步，PI Austin 和合作研究员 Feng 解决了具有结合底物的 AlkB 的第一个结构，并表明它是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，连同建立一种快速功能表征的新测定方法并开发一套 AlkB 活性 AlkB 同源物，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 将整合结构、功能、生物化学、计算和光谱研究以确定二铁活性位点的三维结构，识别底物特异性的决定因素，了解 AlkB 如何被其伴侣蛋白激活，并探究如何激活存在共价结合的电子转移伙伴，仅在一类革兰氏阳性菌中发现细菌，改变该酶家族的反应性。通过这样做，他们将扩展打破和建立关键化学键的策略的基础知识，这可能会导致新的合成路线的开发，以挽救生命和延长生命分子。他们的工作还将为针对该酶家族的努力提供重要的见解治疗目的。