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: 6751919
• 负责人: Antony M. DEAN
• 金额: $ 29.44万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6751919
• 关键词: Escherichia coli; NAD(P)H dehydrogenase; biochemical evolution; catalyst; cofactor; computer graphics /printing; enzyme activity; enzyme structure; enzyme substrate; flow cytometry; isocitrate dehydrogenase; molecular shape; nucleic acid sequence; polymerase chain reaction; protein metabolism; protein purification; protein structure; protein structure function; site directed mutagenesis

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.

描述（申请人的摘要）：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。 通过将酶的结构与酶的功能联系起来，并将酶的功能与 健身，这项工作将提供对健身原因的详细了解 适应和约束在生化进化中的作用。 NADP 依赖性异柠檬酸脱氢酶 (IDH) 和 NAD 依赖性 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。这两种酶都属于 古老而广泛的超家族，其系统发育提供了进化 历史。它们在新陈代谢中的作用已被彻底了解，亮氨酸中的 IMDH 克雷布斯循环中的生物合成和 IDH。它们的动力学、催化和调节 机制（转录水平的 IMDH，翻译后水平的 IDH） 磷酸化）已确定。天然的、突变的和修饰的酶， 有和没有结合底物。它已经接受了详细的X射线检查 晶体学研究 这种丰富而详细的背景为 了解分子进化中的适应和约束。 系统发育分析表明，35 亿年前，一种古老的细菌 NAD 依赖性 IDH 进化出了利用 NADP 的能力。相比之下，大家都知道 IMDH 利用 NAD。蛋白质工程已证实 250 种中只有 6 种 氨基酸替代决定使用哪种辅酶。网站这么少 确定辅酶的使用，以便在 可以构建两种极端的表型。菌株之间的竞争 携带不同突变等位基因的大肠杆菌将用于确定 健身。因此，催化效率、底物之间的关系 特异性和适应性将被严格确定，从而使分子 IDH 辅酶利用适应性转变的基础（用于生长 醋酸盐），以及迫使 IMDH 使用 NAD（具有 中间表型不太适合）从适应性的角度来理解 风景。 通过调查 40 亿年来发生和未发生的事情 分子进化史不仅会丰富我们对 生化适应，但也可能提供对这种关系的微妙见解 蛋白质结构和功能之间的关系，可能被更多人忽视 传统方法。其中许多可能对合理设计有帮助 工业催化剂和医药药物。