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

智力优势：蛋白质是执行细胞、组织和器官工作的分子机器，并最终协调个体的发展。尽管蛋白质在生命生物学中发挥着核心作用，但促进蛋白质功能进化的机制仍不清楚。理想情况下，研究古代生物的蛋白质是为了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，本研究将采用古分子方法复活已灭绝的蛋白质，以研究蛋白质功能进化的历史。在本研究中，将为一组植物代谢物产生酶合成一种3亿年前的祖先蛋白质及其后代，以测试几个假设。要验证的第一个假设是，祖先的酶最初是“万事通”，后来在基因复制后产生了专门的后代。这一假设从未在任何不同的酶家族中进行过测试，因此这一结果将填补理解蛋白质功能多样化模式的空白。第二个有待检验的假设是，存在许多不同的突变路径，使不同的祖先蛋白质进化出相似的功能。在这种情况下，导致咖啡和茶谱系中咖啡因合成活性独立进化的突变路径将被概述。酶活性的趋同进化以前从未在这个水平上进行过研究，因此这项研究将阐明其发生的详细机制。更广泛的影响：这项研究将为植物防御和重要的次生化合物（如咖啡因）的产生提供见解，从而对农业和植物代谢工程具有潜在的影响。此外，这项研究可能对蛋白质工程有启示，因为祖先酶提供了有用的点，开始选择所需的活动，因为它们的广谱能力。本科生和研究生将完成本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说明他们的发现。此外，这里提出的概念问题在高级分子系统发育和进化课程中作为一个主题实施，该课程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。进化的主题也构成了分子生物学实验课程的基础，在这门课程中，通过进行基因克隆、蛋白质表达、酶分析和定点诱变，实验被用来检验与自然选择对蛋白质功能进化的影响有关的假设。因此，拟议的研究和这两门课程为学生提供了一个关于蛋白质进化的综合和概念性的视角，将他们在化学和生物学方面的训练结合起来。

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.