Evolution of a Dehydrogenase in its Adaptive Landscape

描述(由申请人提供)：本项目的目标是确定两种酶在其上进化的适应环境的分子结构。通过将酶结构与酶功能、酶功能与代谢流量、代谢流量与达尔文适合度联系起来，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，一种依赖于NAD的古老细菌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。蛋白质工程学已经证实，在250个氨基酸替代中，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，可以构建这两种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被用来确定适合性。因此，将严格确定催化效率、底物专一性和适合性之间的关系，使异柠檬酸脱氢酶(用于在醋酸盐上生长)对辅酶利用的适应性转变的分子基础，以及迫使异丙基苹果酸脱氢酶使用NAD(中间表型的酶不太适合)的限制因素能够从适应性的角度得到理解。 通过研究在40亿年的分子进化历史中发生了什么，没有发生什么，不仅将丰富我们对生化适应的理解，还可能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传统的方法忽视。其中许多可能被证明有助于工业催化剂的合理设计和药物的合理设计。

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.