Catalytically Promiscuous Enzymes As Evolutionary Starting Points

催化混杂酶作为进化起点

基本信息

批准号：
8085790
负责人：
ICHIRO MATSUMURA
金额：
$ 33.42万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-09-26 至 2013-06-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): My goal is to understand how new catalytic mechanisms evolve. Current biochemistry textbooks emphasize the specificity and efficiency of extant enzymes. The changes in protein structure that create new catalytic activities are difficult to understand, even in retrospect, and nearly impossible to predict or model. My research team used functional genomics tools to identify 41 multi-copy suppressors that rescue 21 Escherichia coli auxotrophs from starvation on minimal media. Most of the selected proteins are unrelated in structure to those missing in the rescued auxotrophs, but some evince broad similarities in catalytic function. These surprising preliminary results suggest that protein multifunctionality is common and biologically relevant. Here I propose to evolve selected promiscuous enzymes in vitro, and to study the resulting changes in active-site structure and catalytic mechanism. These experiments will establish a simple structural model that will facilitate the directed evolution of new protein pharmaceuticals ("biologics") and diagnostic reagents. Public Health Relevance: Protein engineers emulate molecular evolution in the laboratory to create new protein pharmaceuticals and diagnostic reagents. The experiments described in this grant proposal will help us to better understand adaptive evolution at the molecular level, thereby accelerating the artificial evolution of clinically useful biomolecules.

描述（由申请人提供）：我的目标是了解新的催化机制是如何演变的。当前的生物化学教科书强调现有酶的特异性和效率。即使回想起来，产生新催化活性的蛋白质结构变化也很难理解，而且几乎不可能预测或建模。我的研究团队使用功能基因组学工具鉴定了 41 个多拷贝抑制子，这些抑制子可以在基本培养基上拯救 21 个大肠杆菌营养缺陷型。大多数选定的蛋白质在结构上与获救的营养缺陷型中缺失的蛋白质无关，但有些蛋白质在催化功能上表现出广泛的相似性。这些令人惊讶的初步结果表明蛋白质多功能性是常见的并且具有生物学相关性。在这里，我建议在体外进化选定的混杂酶，并研究由此产生的活性位点结构和催化机制的变化。这些实验将建立一个简单的结构模型，促进新型蛋白质药物（“生物制剂”）和诊断试剂的定向进化。公共健康相关性：蛋白质工程师在实验室中模拟分子进化，以创造新的蛋白质药物和诊断试剂。该资助提案中描述的实验将帮助我们更好地理解分子水平上的适应性进化，从而加速临床有用的生物分子的人工进化。