Evolution of a Dehydrogenase in its Adaptive Landscape

脱氢酶在其适应性景观中的进化

• 批准号: 7214828
• 负责人: Antony M. DEAN
• 金额: $ 30.85万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-12-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7214828
• 关键词: Acetates; Active Sites; Alleles; Amino Acids; Anabolism; Biochemical; Citric Acid Cycle; Coenzymes; Complex; Costs and Benefits; Drug Industry; Environment; Enzymes; Escherichia coli; Evolution; Funding; Genetic; Glucose; Goals; Growth; Isocitrate Dehydrogenase; Kinetics; Leucine; Maps; Medicine; Metabolic; Metabolism; Metaphor; Methods; Mitochondria; Molecular; Molecular Structure; Movement; Mutation; NADP; Natural Selections; Nicotinamide adenine dinucleotide; Nutrient; Oxidoreductase; Pathway interactions; Phenotype; Phylogenetic Analysis; Production; Property; Protein Engineering; Recording of previous events; Research; Site; Source; Structure; Substrate Specificity; Testing; Thinking; Today; Work; base; biochemical evolution; catalyst; design; enzyme structure; fitness; insight; mutant; preference; protein structure function

DESCRIPTION (provided by applicant): The goal of this project is to determine the molecular structure of the adaptive landscapes across which two enzymes evolve. By relating enzyme structure to enzyme function, enzyme function to metabolic flux, and metabolic flux to Darwinian fitness, this work will provide a detailed understanding of the causes of adaptation and constraint in biochemical evolution. Phylogenetic analyses reveal that 3.5 billion years ago an ancient bacterial NAD-dependent isocitrate dehydrogenase evolved the ability to utilize NADP. In contrast, all known isopropylmalate dehydrogenases utilize NAD. Protein engineering has confirmed that only 6 out of 250 amino acid replacements determine which coenzyme is used. With so few sites determining coenzyme usage, all possible genetic intermediates between the two extreme phenotypes can be constructed. Competition between strains of Escherichia coli carrying different mutant alleles will be used to determine fitnesses. Thus, the relations between catalytic efficiency, substrate specificity and fitness will be rigorously determined, enabling the molecular basis of the adaptive shift in coenzyme utilization by isocitrate dehydrogenase (for growth on acetate), and the constraints that force isopropylmalate dehydrogenase to use NAD (enzymes with intermediate phenotypes are less fit) to be understood in terms of adaptive landscapes. By investigating what has, and has not, happened during 4 billion years of molecular evolutionary history will not only enrich our understanding of biochemical adaptation, but may also provide subtle insights into the relations between protein structure and function, ones that might be overlooked by more traditional approaches. Many of these may prove helpful to the rational design of catalysts for industry, and of drugs for medicine.

描述（由申请人提供）：该项目的目标是确定两种酶进化的适应性景观的分子结构。通过将酶结构与酶功能、酶功能与代谢流以及代谢流与达尔文适应性联系起来，这项工作将提供对生化进化中适应和限制的原因的详细理解。 系统发育分析表明，35 亿年前，一种古老的细菌 NAD 依赖性异柠檬酸脱氢酶进化出了利用 NADP 的能力。相比之下，所有已知的异丙基苹果酸脱氢酶都利用 NAD。蛋白质工程已证实，250 个氨基酸替换中只有 6 个决定了使用哪种辅酶。由于决定辅酶使用的位点如此之少，因此可以构建两种极端表型之间所有可能的遗传中间体。携带不同突变等位基因的大肠杆菌菌株之间的竞争将用于确定适应性。因此，催化效率、底物特异性和适合度之间的关系将得到严格确定，从而为异柠檬酸脱氢酶（用于在乙酸盐上生长）利用辅酶的适应性转变提供分子基础，以及迫使异丙基苹果酸脱氢酶使用NAD（酶）的约束条件。具有中间表型的不太适合）可以根据适应性景观来理解。 通过研究 40 亿年的分子进化史中发生了什么和没有发生什么，不仅可以丰富我们对生化适应的理解，还可以为蛋白质结构和功能之间的关系提供微妙的见解，而这些关系可能会被更多人忽视。传统方法。其中许多可能有助于工业催化剂和医学药物的合理设计。