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）：该项目的目的是确定 自适应景观的分子结构，两种酶都会发展。 通过将酶结构与酶功能联系起来，并将酶功能与 健身，这项工作将详细了解 约束在生化进化中的适应和作用。 NADP依赖性异位酸脱氢酶（IDH）和NAD依赖性 埃切里希菌线圈的异丙基乳酸脱氢酶（IMDH）提供了理想 探索这些关系的实验系统。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充分理解，在亮氨酸中 Krebs周期中的生物合成和IDH。它们的动力学，催化和调节性 机制（转录级别的IMDH，IDH通过翻译后 磷酸化）已确定。天然，突变和改良的酶， 有和没有底物约束。它受到详细的X射线检查 晶体学研究 这种丰富而详细的背景为 了解分子进化的适应和约束。 系统发育分析表明，有35亿年前的一个古老细菌 NAD依赖性IDH发展了使用NADP的能力。相反，所有已知 IMDHS利用NAD。蛋白质工程已经确认250个中只有6个 氨基酸替代品确定使用了哪种辅酶。很少有网站 确定辅酶使用情况，以便所有可能的遗传中间体 可以构建两个极端表型。菌株之间的竞争 携带不同突变等位基因的大乙烯线圈将用于确定 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，使分子能够 IDH的辅酶利用率自适应转移的基础（用于增长 乙酸盐），以及迫使IMDH使用NAD的约束（酶 中间表型不太适合）可以从自适应方面理解 风景。 通过调查已有40亿年以来没有发生过的情况，没有发生的事情 分子进化史不仅会丰富我们对 生化适应，但也可能为关系提供微妙的见解 在蛋白质结构和功能之间，可能会被更多忽视的蛋白质 传统方法。其中许多可能对理性设计有帮助 用于工业的催化剂和药物的药物。