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

描述(由申请人提供)：项目摘要：自然界利用二铁羧酸酶催化各种氧化反应，包括将甲烷转化为甲醇，形成不饱和脂肪酸，以及产生有利于脱氧核苷酸生物合成的酪氨酸基。然而，它们不同的反应能力掩盖了它们在结构上的相似之处。这一建议试图通过开发一个准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活性的金属-蛋白质相互作用。在计算蛋白质设计方面的进展导致了含有二铁羧酸活性中心(DFsc)的自组装四螺旋束的发展。这种支架非常适合于系统地研究天然二铁羧酸酶中发现的结构/功能关系，因为它很容易通过点突变进行修饰，并且它不仅模拟了天然二铁簇的第一配位球，而且还模拟了第二配位球和第三配位球。DFsc及其变体的结构表征将利用X射线结晶学进行。随后，这些蛋白质的氧化还原电位和催化能力将作为其结构变化的函数进行测定和分析。最后，为了测试我们对天然二铁羧酸盐中结构/功能关系的理解，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：金属酶在生物系统中普遍存在，是无数细胞过程所必需的。这项研究试图在分子水平上理解自然界如何利用金属离子独特的几何和电子性质来进行催化活性。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.