Catalytically Promiscuous Enzymes As Evolutionary Starting Points

描述（由申请人提供）：我的目标是了解新的催化机制是如何发展的。目前的生物化学教科书强调现存酶的特异性和效率。产生新的催化活性的蛋白质结构的变化很难理解，甚至回顾过去，几乎不可能预测或建模。我的研究小组使用功能基因组学工具鉴定了41个多拷贝抑制因子，这些抑制因子在最小的培养基上拯救了21个大肠杆菌营养不良菌。大多数选择的蛋白质在结构上与拯救的营养缺陷细胞中缺失的蛋白质无关，但有些蛋白质在催化功能上有广泛的相似性。这些令人惊讶的初步结果表明，蛋白质的多功能性是普遍的，并且具有生物学相关性。在此，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，以促进新的蛋白质药物（“生物制剂”）和诊断试剂的定向进化。公共卫生相关性：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。本基金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，从而加速临床有用生物分子的人工进化。

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.