De Novo Design of a Catalytically Active Di-Iron Carboxylate Enzyme

项目概述：二铁羧酸酶在自然界中被用来催化多种氧化反应，包括甲烷转化为甲醇，不饱和脂肪酸的形成，以及促进脱氧核糖核苷酸生物合成的酪氨酸自由基的生成。然而，它们不同的反应性掩盖了它们结构上的相似性。本提案旨在通过开发一个精确模拟天然酶的几何、电子和催化性质的模型系统，在分子水平上理解控制二铁羧酸酶反应性的金属-蛋白质相互作用。计算蛋白设计的进步导致了包含二羧酸铁活性位点（DFsc）的自组装四螺旋束的发展。这种支架非常适合系统地研究天然二铁羧酸酶的结构/功能关系，因为它很容易通过点突变进行修改，而且它不仅模仿天然二铁簇的第一配位球，而且还模仿第二和第三配位球。利用x射线晶体学对DFsc及其变体进行结构表征。随后，这些蛋白质的氧化还原电位和催化能力将被确定和分析为其结构变化的函数。最后，作为我们对天然二铁羧酸酯中存在的结构/功能关系的理解的测试，DFsc将通过计算重新设计，以通过稳定假设的催化中间体来提高其催化效率。相关性：金属酶在生物系统中无处不在，是无数细胞过程所必需的。本研究旨在从分子水平上了解大自然如何利用金属离子的独特几何和电子特性来进行催化活性。这些知识可以用来帮助开发仿生催化剂。

DESCRIPTION (provided by applicant): Project Summary: Di-iron carboxylate enzymes are utilized by nature to catalyze a wide variety of oxidation reactions, including the conversion of methane to methanol, the formation of desaturated fatty acids, and the generation of a tyrosyl radical that facilitates the biosynthesis of deoxyribonucleotides. Their divergent reactivities, however, belie their structural similarities. This proposal seeks to comprehend the metal-protein interactions that govern the reactivities of the di-iron carboxylate enzymes at a molecular level by developing a model system which accurately mimics the geometric, electronic, and catalytic properties of the natural enzymes. Advances in computational protein design have led to the development of a self-assembling four-helix bundle that contains a di-iron carboxylate active site (DFsc). This scaffold is well-suited for systematic investigations of the structure/function relationships found in the natural di-iron carboxylate enzymes as it is easily modifiable via point mutations and it mimics not only the first, but also the second and third, coordination spheres of the natural di-iron cluster. Structural characterization of DFsc, and variants will be undertaken utilizing X-ray crystallography. Subsequently, the redox potentials and catalytic abilities of these proteins will be determined and analyzed as a function of their structural variations. Finally, as a test of our understanding of the structure/function relationships present in the natural di-iron carboxylates, DFsc will be computationally redesigned to enhance its catalytic efficiency via stabilization of postulated catalytic intermediates. Relevance: Metalloenzymes are ubiquitous in biological systems and are required for a myriad of cellular processes. This research seeks to understand on a molecular level how nature harnesses the unique geometric and electronic properties of metal ions for catalytic activity. This knowledge can then be used to aid in the development of biomimetic catalysts.