Development of a Novel Artificial Diiron Protein with N-hydroxylase Activity

具有 N-羟化酶活性的新型人工二铁蛋白的开发

• 批准号: 8004780
• 负责人: Marcos M. Pires
• 金额: $ 4.1万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8004780
• 关键词: Active Sites; Affect; Anabolism; Antibiotics; Binding; Biological Process; Carrier Proteins; Complex; Crystallography; Development; Dioxygen; Elements; Environment; Enzymes; Family member; Ferritin; Goals; Heme; Housing; Hydrogenase; Ligand Binding; Ligands; Measurement; Measures; Metabolic; Metal Binding Site; Metals; Methane hydroxylase; Methods; Mixed Function Oxygenases; Modeling; Mutate; Mutation; Protein Family; Proteins; Reaction; Ribonucleotide Reductase; aureothin; desaturase; inositol oxygenase; insight; member; novel; public health relevance

DESCRIPTION (provided by applicant): Non-heme diiron enzymes represent a diverse class of proteins that are responsible for a variety of biological functions by binding and activating dioxygen. Some of the members of this growing family of proteins include methane monooxygenase, ribonucleotide reductase, stearoylacyl carrier protein d9-desaturase, myo-inositol oxygenase, and ferritins. While the binuclear metal binding sites among most of the members of this family are composed of the same 4 Glu and 2 His ligand set, they perform dissimilar reactions. How these proteins are able to perform such diverse tasks using similar active sites remains a challenging question. The answer to this question will help us understand essential details of how the environment surrounding the active site acts to tune and modulate the reactivity of the binuclear metal binding sites. To this end, minimal model proteins that incorporate elements that are responsible for reactivity have generated significant insight into the relationship between the protein environment and the reactivity of diiron proteins. They possess the ability to house an active site within the core of a 4 helix bundle protein, yet are small enough that they are readily expressed, mutated, and studied using a number of structural and spectroscopic methods. Here, a novel artificial non-heme diiron protein that mimics the unusual protein AurF is described. AurF is a recently discovered N-hydrogenase involved in the biosynthesis of the polyketide antibiotic aureothin. When the metal binding site of AurF (4 Glu/3 His) was installed into the previously constructed artificial minimal diiron enzymes, it was found to also have N-hydrogenase activity. The goal of this project will be to understand how the environment surrounding the metal binding sites affects the N-hydrogenase activity of the novel DFscH3 model protein. Structural and spectroscopic measurements will be used to measure the changes in the metal ligand environment and its reactivity upon the creation of new constructs with mutations in the second/third ligand shell, metal binding ligand set, and the substrate access channel. The information gained from these studies will be correlated to how natural non-heme diiron enzymes use the protein environment to induce different reactivity from similarly built metal binding sites. PUBLIC HEALTH RELEVANCE: Non-heme diiron proteins are essential enzymes that catalyze a variety of reactions through the activation of dioxygen and they are involved in a wide range of metabolic functions. Minimal model proteins that mimic the much larger and complex natural proteins can be extremely useful in deciphering the elements responsible for the enzyme reactivity. Here we seek to explore an artificial minimal model of the naturally occurring protein AurF, which is involved in the biosynthesis of the polyketide antibiotic aureothin.

描述（由申请人提供）：非血红素二铁酶代表了通过结合和激活分子氧负责多种生物学功能的多种蛋白质。这个不断增长的蛋白质家族的一些成员包括甲烷单加氧酶、核糖核苷酸还原酶、硬脂酰酰基载体蛋白d9-去饱和酶、肌醇加氧酶和铁蛋白。虽然这个家族的大多数成员中的双核金属结合位点由相同的4个Glu和2个His配体组组成，但它们进行不同的反应。这些蛋白质如何能够使用相似的活性位点执行如此不同的任务仍然是一个具有挑战性的问题。这个问题的答案将帮助我们了解活性位点周围的环境如何调节和调节双核金属结合位点的反应性的基本细节。为此，最小的模型蛋白，纳入负责反应性的元素产生了显着的洞察蛋白质环境和二铁蛋白的反应性之间的关系。它们具有在4螺旋束蛋白的核心内容纳活性位点的能力，但足够小，使得它们容易表达、突变，并使用许多结构和光谱方法进行研究。在这里，一种新的人工非血红素二铁蛋白，模仿不寻常的蛋白质AuRF的描述。AurF是最近发现的参与聚酮抗生素aureothin生物合成的N-氢化酶。当AuRF的金属结合位点（4 Glu/3 His）被安装到先前构建的人工最小二铁酶中时，发现它也具有N-氢化酶活性。该项目的目标是了解金属结合位点周围的环境如何影响新型DFscH 3模型蛋白的N-氢化酶活性。结构和光谱测量将用于测量金属配体环境的变化及其在第二/第三配体壳、金属结合配体组和底物进入通道中具有突变的新构建体产生后的反应性。从这些研究中获得的信息将与天然非血红素二铁酶如何使用蛋白质环境来诱导来自类似构建的金属结合位点的不同反应性相关。 公共卫生相关性：非血红素二铁蛋白是通过激活分子氧催化多种反应的必需酶，它们参与广泛的代谢功能。模拟更大和复杂的天然蛋白质的最小模型蛋白质在破译负责酶反应性的元素方面非常有用。在这里，我们试图探索一个人工的最小模型的天然存在的蛋白质AurF，这是参与生物合成的聚酮抗生素aureothin。