Evolutionary Insights Into Enzyme Mechanisms

对酶机制的进化见解

• 批准号: 8690916
• 负责人: Antony M. DEAN
• 金额: $ 43.95万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8690916
• 关键词: Active Sites; Alanine Racemase; Amino Acids; Binding; Biochemistry; Bioinformatics; Biology; Bioremediations; Carbon; Catalysis; Chemistry; Chocolate; Coenzymes; Data; Disease; Distant; Drug Design; Drug Industry; Engineering; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzyme Kinetics; Enzymes; Evolution; Family; Future; Genes; Genetic Epistasis; Glycine Hydroxymethyltransferase; Goals; HIV Protease Inhibitors; Health; Human; Hydrolase; Inborn Genetic Diseases; Industry; Isocitrate Dehydrogenase; Knowledge; Learning; Life; Lyase; Metabolism; Methodology; Methods; Molecular Biology; Mutagenesis; Mutation; Pharmaceutical Preparations; Pharmacologic Substance; Phylogenetic Analysis; Pollution; Production; Pyridoxal Phosphate; Reaction; Recreation; Research; Research Personnel; Site; Site-Directed Mutagenesis; Solutions; Specialist; Specificity; Stem cells; Testing; Work; base; catalyst; chemical reaction; design; directed evolution; drug synthesis; enzyme mechanism; esterase; gene synthesis; insight; interdisciplinary approach; knowledge base; novel; novel strategies; progenitor; protein folding; reconstruction; screening; success; theories

DESCRIPTION (provided by applicant): Enzymes catalyze the reactions essential to biology. In broad strokes, researchers understand how they work, but not how they work well. The complexity of enzymes makes it hard to identify which parts are important to catalysis. The amino acids in the reaction site are essential, but not enough to create highly active enzymes. Distant residues are also important and some residues are helpful only in some cases, when other residues are also present (epistasis). Identifying all residues that contribute to catalysis s an essential first step to learn why enzymes work well. To identify these residues, we propose to recreate ancestral enzymes. This recreation reduces complexity because it focuses on those few mutations directly associated with historical functional changes, thereby avoiding the pitfalls inherent to using (incomplete) knowledge based approaches. The number of differences that need to be screened is reduced from hundreds to tens enabling the systematic use of site directed mutagenesis to explore the contribution that each mutation makes to changes in function. Ancestral sequence reconstruction also has the potential of identify epistatic sites that form local optima on which directed evolution methods get trapped. Parallel acquisitions and reversals in function within superfamilies allow alternative solutions to be explored. Preliminary data show that the approach is feasible: ancestral enzymes have been synthesized, shown to be active and more promiscuous than modern specialist descendants and sites critical to high activity outside active sites have been found. Recreating catalytically promiscuous ancestral "stem cell" enzymes allows us to recapture the functional plasticity needed to identify what determines different types of activity in the same protein fold.

描述（由申请人提供）：酶催化对生物学必不可少的反应。在广泛的笔触中，研究人员了解他们的工作原理，但他们的工作方式不佳。酶的复杂性使难以确定哪些部分对催化很重要。反应位点中的氨基酸是必不可少的，但不足以产生高度活性的酶。遥远的残留物也很重要，只有在某些情况下也存在其他残留物（epitsasis），有些残基才有帮助。识别所有有助于催化的残留物是了解为什么酶正常工作的重要第一步。为了识别这些残基，我们建议重新创建祖先酶。这种娱乐活动降低了复杂性，因为它专注于与历史功能变化直接相关的少数突变，从而避免了陷阱 使用（不完整）基于知识的方法固有。需要筛选的差异数量从数百个减少到数十个，从而可以系统地使用定向诱变，以探索每个突变对功能变化的贡献。祖先序列重建也有可能识别上位的位点 形成局部最佳选择，将定向演化方法捕获。超家族中功能中功能中的并行采集和逆转允许探索替代解决方案。初步数据表明，该方法是可行的：与现代专业的后代相比，已经合成了祖先酶，表现为活跃和更混乱，并且已经发现了对高活动现场活跃地点至关重要的地点。重现催化杂乱的杂物“干细胞”酶使我们能够重新恢复所需的功能可塑性，以确定在同一蛋白质折叠中确定不同类型活性的方法。