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

• 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
• 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

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.

项目摘要 许多维持生命的过程需要与大气氧的反应。 III级二龙 蛋白质使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都起着重要作用 和新陈代谢，并与人类健康问题有关，包括肥胖，糖尿病，注意力缺陷 障碍和神经变性。它们对于石油的自然生物修复也至关重要。有一个 关于这个膜酶家族的机械信息的缺乏，主要是因为它们 膜相关的性质使它们很难净化和研究。烷烃单加氧酶 （ALKB）是III类积分膜二氮蛋白以及脂肪酸去饱和酶的成员 和脂肪酸羟化酶。 ALKB的氨基酸序列表明它在结构上并不相似 具有相似功能的其他酶。确定其三维结构是一项壮举 数十年来一直躲避科学家。 在初步工作的重要一步中，Pi Austin和共同投资者Feng解决了 ALKB具有绑定底物的第一结构，并表明它是一个出色的模型系统 了解III类二铁蛋白的催化机制。这个突破，以及 建立一种新颖的测定法，用于快速功能表征和开发一套 ALKB活跃的ALKB同源物，为回答有关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，生化，计算和 光谱研究以确定二铁主动部位的三维结构，确定 底物特异性的决定因素，了解其伴侣蛋白如何激活ALKB，然后探测如何 共价结合的电子转移伙伴的存在，仅在一类革兰氏阳性中发现 细菌，改变了该酶家族的反应性。 这样一来，他们将扩大有关破坏和建立关键化学债券的策略的基本知识， 这可能导致发展新的合成路线，以挽救生命和延长生命 分子。他们的工作还将为努力瞄准这个酶家族的努力提供关键的见解 治疗目的。