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(申请人提供)：酶催化生物学必需的反应。总的来说，研究人员了解它们是如何工作的，但不知道它们是如何工作得很好的。酶的复杂性使人们很难确定哪些部分对催化很重要。反应部位的氨基酸是必需的，但不足以产生高活性的酶。远距离残基也很重要，一些残基只有在某些情况下才有帮助，当其他残基也存在时(上位性)。确定所有有助于催化的残基S是了解酶为什么工作得很好的必要的第一步。为了鉴定这些残基，我们建议重建祖先的酶。这种重构降低了复杂性，因为它集中在与历史功能变化直接相关的少数几个突变上，从而避免了陷阱 使用(不完整的)基于知识的方法所固有的。需要筛选的差异从数百个减少到几十个，从而能够系统地使用定点突变来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位点， 形成局部最优，定向进化方法在该局部最优解上陷入陷阱。超级家族内部的平行收购和功能逆转允许探索替代解决方案。初步数据表明，这种方法是可行的：已经合成了祖先的酶，显示出比现代专家后代更有活性和更多的混杂，并发现了活性位点以外对高活性至关重要的位点。通过重新创造催化混杂的祖先“干细胞”酶，我们可以重新获得识别同一蛋白质折叠中决定不同类型活动的因素所需的功能可塑性。