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

催化活性二铁羧酸酶的从头设计

• 批准号: 7276850
• 负责人: Amanda J. Reig
• 金额: $ 4.48万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7276850
• 关键词: Active Sites; Amino Acid Sequence; Anabolism; Biological; Biological Models; Biomimetics; Catalytic Domain; Cell physiology; Classification; Complex; Data; Deoxyribonucleotides; Development; Dioxygen; Electronics; Environment; Enzymes; Family; Family member; Fatty Acids; Generations; Goals; Helix (Snails); Histidine; Hydrocarbons; Hydroxylation; Investigation; Ions; Iron; Knowledge; Ligands; Metal Binding Site; Metals; Methane; Methane hydroxylase; Methanol; Methods; Modeling; Molecular; Nature; Oxidation-Reduction; Peptide Sequence Determination; Personal Satisfaction; Point Mutation; Positioning Attribute; Property; Proteins; Reaction; Research; Series; Solubility; Solutions; Structure; Structure-Activity Relationship; System; Testing; Thinking; Variant; X-Ray Crystallography; aqueous; carboxylate; catalyst; desaturase; design; ear helix; insight; metalloenzyme; oxidation; ribonucleotide reductase M2; ribonucleotide reductase R2 subunit; scaffold

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.

描述（由申请人提供）：项目摘要：二氨基羧酸酯酶自然地催化了多种氧化反应，包括甲烷转化为甲醇，脱饱和脂肪酸的形成以及酪酰基的产生，从而促进了脱氧核酸脂蛋白的生物合成的生物合成。然而，他们的不同反应性是他们的结构相似性。该提案旨在通过开发一个模型系统来准确地模拟天然酶的几何，电子和催化性能，从而理解分子水平上Di-Iron羧酸盐酶的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一个自组装的四螺旋束的发展，该束包含Di-Iron羧酸盐活性位点（DFSC）。该脚手架非常适合系统地研究天然Di-Iron羧酸盐酶中发现的结构/功能关系，因为它可以通过点突变很容易修改，并且不仅模拟了第一个和第三个和第三，也可以模仿自然二型铁簇的第二和第三个配位球。 DFSC的结构表征和变体将使用X射线晶体学进行。随后，将确定和分析这些蛋白质的氧化还原电位和催化能力，这是其结构变化的函数。最后，作为我们对天然Di-Iron羧酸盐中存在的结构/功能关系的理解，DFSC将在计算上进行重新设计，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，并且需要无数的细胞过程。这项研究试图在分子水平上理解自然如何利用金属离子的独特几何和电子特性来催化活性。然后，这些知识可以用来帮助开发仿生催化剂。