Evolution of a Dehydrogenase in its Adaptive Landscape

描述(申请人摘要)：本项目的目标是确定 两种酶在其中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，将酶功能与 健身，这项工作将提供一个详细的原因的了解 适应和限制在生化进化中的作用。 依赖NADP的异柠檬酸脱氢酶(IDH)和依赖NAD的 大肠杆菌的异丙基苹果酸脱氢酶(IMDH)提供了理想的 实验系统来探索这些关系。两种酶都属于一种 古老而广泛的超级家族，其发展史为进化提供了 历史。它们在新陈代谢中的作用已被彻底了解，亮氨酸 克雷布斯循环中的生物合成和IDH。它们的动力学、催化和调控 机制(转录水平的IMDH，翻译后的IDH 磷酸化)已被测定。天然的、突变的和修饰的酶， 结合和不结合底物。它已经接受了详细的X光检查 结晶学研究 这一丰富而详细的背景为 理解分子进化中的适应和约束。 系统发育分析显示，35亿年前，一种古老的细菌 依赖NAD的IDH进化出利用NADP的能力。相比之下，大家都知道 IMDH利用NAD。蛋白质工程学已经证实，250人中只有6人 氨基酸替代决定使用哪种辅酶。只有这么少的网站 确定辅酶的使用，以便所有可能的遗传中间体 可以构建两种极端的表型。不同菌株之间的竞争 携带不同突变等位基因的大肠杆菌将被用来确定 健身。因此，催化效率、底物之间的关系 特异性和适合性将被严格确定，使分子 IDH对辅酶利用的适应性转变的基础(用于生长 醋酸盐)，以及迫使IMDH使用NAD(酶与 中间表型不太适合)从适应性的角度来理解 风景画。 通过调查40亿年来发生和没有发生的事情 分子进化史不仅将丰富我们对 生化适应，但也可能提供对这些关系的微妙见解 在蛋白质结构和功能之间，可能被更多人忽视的问题 传统的方法。其中许多可能被证明对合理的设计有帮助 用于工业的催化剂和用于医药的药物。

DESCRIPTION (Applicant's Abstract):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, and enzyme function to fitness, this work will provide a detailed understanding of the causes of adaptation and role of constraint in biochemical evolution. The NADP-dependent isocitrate dehydrogenase (IDH) and NAD-dependent isopropylmalate dehydrogenase (IMDH) of Escherichia coil provide an ideal experimental system to explore these relations. Both enzymes belong to an ancient and extensive superfamily, the phylogeny of which provides evolutionary history. Their roles in metabolism are thoroughly understood, IMDH in leucine biosynthesis and IDH in Krebs' cycle. Their kinetic, catalytic and regulatory mechanisms (IMDH at the transcriptional level, IDH by post-translational phosphorylation) have been determined. Native, mutant, and modified enzymes, with and without substrates bound. It had been subject to detailed X-ray crystallographic studies This rich and detailed background provides the necessary basis for understanding adaptation and constraint in molecular evolution. Phylogenetic analyses reveal that 3.5 billion years ago an ancient bacterial NAD-dependent IDH evolved the ability to utilize NADP. In contrast, all known IMDHs 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 so all possible genetic intermediates between the two extreme phenotypes can be constructed. Competition between strains of Escherichia coil carrying different mutant alleles will be used to determine the fitness. 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 IDH (for growth on acetate), and the constraints that force IMDH 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.