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， 健身，这项工作将提供一个详细的了解的原因， 适应和限制在生物化学进化中的作用。 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，其遗传提供了进化的 历史它们在代谢中的作用已被彻底了解，亮氨酸中的IMDH 生物合成和异脱氢酶。它们的动力学、催化和调节作用 转录水平的IMDH，翻译后水平的IDH， 磷酸化）。天然的、突变的和修饰的酶， 结合或不结合底物。它已经接受了详细的X光检查 晶体学研究 这一丰富而详细的背景为以下方面提供了必要的基础： 理解分子进化中的适应和限制。 系统发育分析显示35亿年前一种古老的细菌 NAD依赖性IDH进化出利用NADP的能力。相比之下，所有已知的 IMDHs使用NAD。蛋白质工程已经证实250个中只有6个 氨基酸替代决定了使用哪种辅酶。只有这么少的地点 确定辅酶的使用，使所有可能的遗传中间体之间的 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 特异性和适应性将被严格确定，使分子 IDH在辅酶利用方面的适应性转变的基础（对于生长在 乙酸），以及迫使IMDH使用NAD（具有 中间表型不太适合）从适应性角度理解 的风景. 通过调查在40亿年的时间里发生了什么， 分子进化史不仅会丰富我们对 生化适应，但也可能提供微妙的见解的关系， 蛋白质结构和功能之间的联系， 传统的方法。其中许多可能有助于合理设计 工业催化剂和医药药物。

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.