Ghosts of Evolution Past: Resurrecting an Extinct Ancestral Enzyme to Understand the Origins of Modern-day Biochemical Activities

智力优势：蛋白质是分子机器，它执行细胞、组织和器官的工作，并最终协调个人的发展。尽管蛋白质在生命生物学中扮演着核心角色，但促进蛋白质功能进化的机制仍不清楚。理想的情况是，研究古代生物的蛋白质，以便了解功能如何随着时间的推移而变化，以及相同的功能如何在不同的蛋白质中独立进化。由于古代生物已经灭绝，这项研究将使用古分子方法来复活灭绝的蛋白质，以研究蛋白质功能进化史。在这项研究中，将为一组产生植物代谢物的酶合成一种3亿年前的祖先蛋白质及其后代，以检验几个假设。第一个要检验的假设是，祖先的酶最初是通才的“万能”，后来在基因复制后产生专门的后代。这一假说从来没有对任何不同的酶家族进行过测试，因此，结果将填补理解蛋白质功能多样化模式的空白。需要检验的第二个假设是，存在许多不同的突变路径，允许不同的祖先蛋白质进化出相似的功能。在这种情况下，导致咖啡因合成活性在咖啡和茶叶谱系中独立进化的突变路径将被概括。酶活性的收敛进化以前从未在这个水平上被研究过，所以这项研究将阐明它发生的详细机制。广泛的影响：这项研究将提供对植物防御和重要的次生化合物如咖啡因的产生的洞察，从而对农业和植物代谢工程具有潜在的意义。此外，这项研究可能对蛋白质工程有影响，因为祖先酶提供了有用的点，从那里启动对理想活动的选择，因为它们具有广泛的能力。本科生和研究生将完成这项研究所需的所有实验室实验，并在会议上展示他们的结果，并以手稿的形式详细介绍他们的发现。此外，这里提出的概念问题作为高级分子系统发育和进化课程的一个主题实施，在该课程中，提出了蛋白质进化的适应性假设，并设计了统计测试来测试预测。进化主题也构成了配套的分子生物学实验室课程的基础，在该课程中，实验将通过进行基因克隆、蛋白质表达、酶分析和定点突变来测试与自然选择对蛋白质功能进化的影响有关的假说。因此，拟议的研究和这两门课程为学生提供了关于蛋白质进化的综合和概念性观点，将他们在化学和生物方面的培训结合在一起。

Intellectual Merit: Proteins are molecular machines that carry out the work of cells, tissues and organs and ultimately orchestrate the development of individuals. In spite of the central role proteins play in the biology of life, the mechanisms promoting the evolution of protein functions remain unclear. Ideally, proteins of ancient organisms would be studied in order to understand how function changes over time and how the same function can evolve independently in distinct proteins. Because ancient organisms are extinct, this study will use a paleomolecular approach to resurrect extinct proteins to study the history of protein functional evolution. For this research, a 300 million year old ancestral protein and its descendants will be synthesized for a group of plant metabolite-producing enzymes in order to test several hypotheses. The first hypothesis to be tested is that the ancestral enzymes begin as generalist "jack-of-all trades" and later give rise to specialized descendants after gene duplication. This hypothesis has never been tested for any diverse enzyme family and thus the results will fill a void in understanding the pattern of protein functional diversification. The second hypothesis to be tested is that numerous distinct mutational paths exist which allow different ancestral proteins to evolve similar functions. In this case, the mutational paths leading to the independent evolution of caffeine synthesis activity in the coffee and tea lineages will be recapitulated. Convergent evolution of enzyme activity has never been investigated at this level before so this study will illuminate the detailed mechanism by which it occurs.Broader impacts: This study will provide insight into plant defense and the production of important secondary compounds like caffeine, thereby having potential implications for agriculture and metabolic engineering of plants. In addition, this research may have implications for protein engineering because ancestral enzymes provide useful points from which to initiate selection for desirable activities because of their broad spectrum of capabilities. Undergraduate and graduate students will perform all of the laboratory experiments required for this study and present their results at conferences and as manuscripts detailing their findings. Also, the conceptual issues presented here are implemented as one theme in a senior-level Molecular Phylogenetics and Evolution course in which adaptive hypotheses for protein evolution are proposed and statistical tests are designed to test the predictions. An evolutionary theme also forms the foundation of a companion Molecular Biology Laboratory course in which experiments are used to test hypotheses related to the effects of natural selection on protein functional evolution by performing gene cloning, protein expression, enzyme assays, and site-directed mutagenesis. As such, the proposed research and these two courses provide students with an integrated and conceptual perspective on protein evolution that unites their training in chemistry and biology.