Catalytically Promiscuous Enzymes As Evolutionary Starting Points

催化混杂酶作为进化起点

• 批准号: 7692247
• 负责人: ICHIRO MATSUMURA
• 金额: $ 34.1万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-26 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7692247
• 关键词: Active Sites; Alanine Racemase; Amino Acid Substitution; Applications Grants; Biochemical; Biochemistry; Collection; Cysteine Desulfhydrase; Diagnostic; Diagnostic Reagent; Enzymes; Escherichia coli; Event; Evolution; Gene Deletion; Genes; Goals; In Vitro; Industry; Laboratories; Medicine; Modeling; Molecular; Molecular Evolution; Mutagenesis; Mutation; Open Reading Frames; Outcome; Pharmacologic Substance; Process; Property; Protein Biochemistry; Protein Engineering; Proteins; Public Health; Pyridoxal Phosphate; Reaction; Research; Specificity; Starvation; Structural Models; Structure; Textbooks; Transferase; Variant; Work; base; directed evolution; expression vector; fitness; functional genomics; improved; phosphoribosylanthranilate isomerase; protein structure; public health relevance; research study; therapeutic protein; tool

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(申请人提供)：我的目标是了解新的催化机制是如何演变的。目前的生物化学教科书强调现有酶的特异性和有效性。产生新的催化活性的蛋白质结构的变化很难理解，即使回想起来也是如此，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个多拷贝抑制子，这些抑制子在最低限度的培养基上将21个大肠杆菌营养缺陷症从饥饿中拯救出来。大多数选定的蛋白质在结构上与被拯救的营养缺陷体中缺失的蛋白质无关，但也有一些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，蛋白质的多功能是常见的，并且具有生物学意义。在这里，我建议在体外进化选定的杂交酶，并研究由此导致的活性部位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，促进新的蛋白质药物(“生物制品”)和诊断试剂的定向进化。与公共健康相关：蛋白质工程师在实验室模拟分子进化，创造新的蛋白质药物和诊断试剂。这项拨款提案中描述的实验将帮助我们在分子水平上更好地理解适应性进化，从而加速临床有用的生物分子的人工进化。