Evolution of a Dehydrogenase in its Adaptive Landscape

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、酶功能与代谢通量、代谢通量与达尔文适应性联系起来，这项工作将提供对生物化学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。相反，所有已知的异丙基苹果酸脱氢酶都利用NAD。蛋白质工程已经证实，250个氨基酸中只有6个决定使用哪种辅酶。由于决定辅酶使用的位点如此之少，两种极端表型之间所有可能的遗传中间体都可以构建。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合度。因此，催化效率，底物特异性和健身之间的关系将被严格确定，使辅酶利用异柠檬酸脱氢酶（生长在乙酸）的适应性转变的分子基础，和限制，迫使异丙基苹果酸脱氢酶使用NAD（酶与中间表型不太适合）被理解的适应性景观。 通过研究在40亿年的分子进化史中发生了什么和没有发生什么，不仅会丰富我们对生物化学适应的理解，而且还可能为蛋白质结构和功能之间的关系提供微妙的见解，这些关系可能被更传统的方法所忽视。其中许多可能有助于工业催化剂和药物的合理设计。

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.