Evolution of enzyme structure and function viewed at atomic resolution

描述(申请人提供)：这项研究的目的是探索蛋白质在苹果酸和乳酸脱氢酶(MdHS和LDH，M/LDH家族)中进化出新功能的动态结构机制。我们计划复活祖先M/LDH酶的整个进化谱系，探索它们的生化功能，用X射线结晶学解决它们的结构，并将沿着这些进化轨迹观察到的结构变化与功能变化联系起来。我们的模型系统是苹果酸和乳酸脱氢酶超家族，它包含一些已知的最容易结晶的蛋白质。这两种酶在生命中广泛分布。MDH存在于柠檬酸循环中，催化苹果酸转化为草酰乙酸酯，而LDH将糖酵解的最终产物丙酮酸转化为乳酸。该家族的进化伴随着许多有趣和重要的功能创新，包括底物专一性的急剧变化，嗜热和嗜冷稳定性的获得，通过小效应分子控制变构的获得，以及通过新的蛋白质-蛋白质界面实现同源多聚体。其具体目的是：1)研究MDH和LDH蛋白的进化史，沿多个轨迹重建祖先蛋白序列，并在实验室中恢复它们(利用系统发育推断和人工基因合成)。特别令人感兴趣的是包含重大进化事件的祖先蛋白质，如特异性、催化速率、温度稳定性、变构和寡聚化状态的变化。2)用X射线结晶学方法结晶并测定了MDH和LDH谱系的高分辨结构。分析观察到的结构变化的演变。3)对复活的蛋白质进行功能表征，并将进化路径上的功能变化与其伴随的结构扰动联系起来。为了确定祖先中哪些变化在功能上是重要的，关键残基将被突变并进行生化表征。通过研究这些祖先蛋白及其突变体，我们将经验性地解决分子生物学和进化论中许多悬而未决的问题。活性部位远端的替换如何影响活性？与我们在现代酶中设计的人工突变相比，历史上的氨基酸替代对酶的结构和功能有不同的影响吗？突变之间的相关性(上位性)的重要性是什么？在进化过程中，特异度会增加吗？MDH和LDH的共同祖先是混杂的多功能蛋白吗？这项研究将提供第一张动态的、高分辨率的图片，说明新的生物分子功能是如何在进化过程中通过突变和基因复制产生的。由此产生的数据将有助于回答几个长期存在的进化问题，并将支持我们关于酶功能如何被合理设计的知识。 与公共健康相关：这项提案将探索蛋白质在一类具有医学重要性的酶--苹果酸和乳酸脱氢酶(MDHS和LDHs)中获得新功能的动态进化机制。由于其与人类MDH酶的显著不同，许多细菌MDH酶是重要的潜在药物靶点，包括人类病原体，如疟疾、炭疽病、类杆菌和假单胞菌。这项研究的结果将有助于回答几个长期存在的进化问题，并有助于我们理解酶的功能是如何被工程实现的。

DESCRIPTION (provided by applicant): The goal of this research is to explore the dynamic structural mechanisms by which proteins have evolved novel functions in an important class of enzymes, the malate and lactate dehydrogenases (MDHs and LDHs, M/LDH family). We plan to resurrect entire evolutionary lineages of ancestral M/LDH enzymes, probe their biochemical functions, solve their structures by X-ray crystallography, and correlate the functional changes with structural changes observed along these evolutionary trajectories. Our model system is the malate and lactate dehydrogenase superfamily, which contains some of the most readily crystallizable proteins known. Both enzymes are widely distributed throughout life. MDH is found in the citric acid cycle and catalyzes the interconversion of malate to oxaloacetate, while LDH converts pyruvate, the final product of glycolysis, to lactate. The evolution of this family has been accompanied by many interesting and important functional innovations, including sharp changes in substrate specificity, acquisition of thermophilic and cryophilic stability, and gain of allosteric control by small effector molecules, and homo-multimerization via new protein- protein interfaces. The specific aims are to: 1) Investigate the evolutionary history of MDH and LDH proteins, reconstruct ancestral protein sequences along multiple trajectories, and resurrect them in the lab (using phylogenetic inference and artificial gene synthesis). Of exceptional interest are ancestral proteins bracketing significant evolutionary events, such as changes in specificity, catalytic rate, temperature stability, allostery, and oligomerization state. 2) Crystallize and determine the high-resolution structures of MDH and LDH lineages by X-ray crystallography. Analyze the evolution of observed structural changes. 3) Functionally characterize the resurrected proteins and correlate the functional changes along an evolutionary pathway with their concomitant structural perturbations. To determine which changes in the ancestors are functionally important, key residues will be mutated and biochemically characterized. By investigating these ancestral proteins and their mutants, we will empirically address many unanswered questions in molecular biology and in evolutionary theory. How do substitutions distal from the active site affect activity? Do historical amino acid substitutions have different effects on enzyme structure and function compared to the artificial mutations we engineer in modern enzymes? What is the importance of correlations among mutations (epistasis)? Does specificity increase during evolution? Was the common ancestor of MDH and LDH a promiscuous multifunctional protein? This study will provide the first dynamic, high-resolution picture of how novel biomolecular functions are generated during evolution by mutations and gene duplications. The resulting data will help answer several long-standing evolutionary questions and will bolster our knowledge of how enzymatic functions can be rationally engineered. PUBLIC HEALTH RELEVANCE: This proposal will explore the dynamic evolutionary mechanisms by which proteins have gained novel functions in a medically important class of enzymes, the malate and lactate dehydrogenases (MDHs and LDHs). Due to their significant differences from the human MDH enzymes, many of the bacterial MDH enzymes are important potential drugs targets, including human pathogens such as malaria, anthrax, Bacteroides, and Pseudomonas. The results of this research will aid in answering several long-standing evolutionary questions and will aid our understanding of how enzymatic functions can be engineered.