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

描述(由申请人提供)：非血红素二铁酶代表一种不同类别的蛋白质，通过结合和激活氧气来负责各种生物功能。这个不断增长的蛋白质家族中的一些成员包括甲烷单加氧酶、核苷酸还原酶、硬脂酰基载体蛋白D9-去饱和酶、肌醇加氧酶和铁蛋白。虽然该家族大多数成员之间的双核金属结合位点由相同的4个Glu和2个His配体组成，但它们执行不同的反应。这些蛋白质如何能够使用相似的活性位点执行如此多样化的任务仍然是一个具有挑战性的问题。这个问题的答案将有助于我们理解活性中心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，包含负责反应性的元素的最小模型蛋白质已经对蛋白质环境和双铁蛋白的反应性之间的关系产生了重要的洞察。它们具有在4螺旋束蛋白的核心中容纳活性位点的能力，但又足够小，很容易表达、突变，并使用许多结构和光谱方法进行研究。在这里，描述了一种新的人造非血红素双铁蛋白，它模仿了不寻常的蛋白质AurF。AurF是新近发现的一种N-氢酶，参与聚酮类抗生素金黄色的生物合成。将AurF(4 Glu/3 His)的金属结合部位与先前构建的人工微量双铁酶结合，发现其也具有N-氢酶活性。该项目的目标将是了解金属结合位点周围的环境如何影响新型DFscH3模型蛋白的N-氢酶活性。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/第三配体外壳、金属结合配体集和底物访问通道发生突变的新构建物创建时的反应活性。从这些研究中获得的信息将与天然非血红素双铁酶如何利用蛋白质环境从类似构建的金属结合部位诱导不同的反应相关联。 公共卫生相关性：非血红素二铁蛋白是通过激活氧气来催化各种反应的基本酶，它们参与了广泛的代谢功能。模拟更大更复杂的天然蛋白质的最小模型蛋白质在破译负责酶反应的元件方面非常有用。在这里，我们试图探索一种人工最小模型的自然存在的蛋白质AurF，它参与了聚酮抗生素金黄的生物合成。

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.