Evolution of a Dehydrogenase in its Adaptive Landscape

脱氢酶在其适应性景观中的进化

基本信息

批准号：
7618541
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
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-06-01 至 2011-04-30
项目状态：
已结题

项目摘要

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）：该项目的目的是确定两个酶进化的适应性景观的分子结构。通过将酶结构与酶功能联系起来，酶功能与代谢通量以及代谢通量与达尔文的适应性相关联，这项工作将详细了解生化进化中适应和约束的原因。系统发育分析表明，有35亿年前的一个古老的细菌NAD依赖性异位酸脱氢酶进化了使用NADP的能力。相比之下，所有已知的异丙胺脱氢酶均利用NAD。蛋白质工程已经证实，在250个氨基酸替代品中，只有6个确定使用了哪种辅酶。由于很少有确定辅酶使用的位点，因此可以构建两个极端表型之间的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健身。因此，将严格确定催化效率，底物特异性和适应性之间的关系，从而使等异戊酸酯脱氢酶在辅酶利用中自适应转移的分子基础（用于乙酸的生长），以及迫使异丙醇脱氢酶与eNHAD的限制（均不适合脱氢）的互联剂（均不适合）（均不适合）。自适应景观。通过调查在40亿年的分子进化史中发生的情况，没有发生过的事情，不仅会丰富我们对生化适应的理解，而且还可以提供对蛋白质结构与功能之间关系的微妙见解，蛋白质结构和功能之间的关系可能会被更多传统的方法所忽视。其中许多可能证明对工业催化剂的合理设计和医学药物的理性设计有帮助。