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

过去进化的幽灵：复活一种已灭绝的祖先酶以了解现代生化活动的起源

• 批准号: 1120624
• 负责人: Todd Barkman
• 金额: $ 26.42万
• 依托单位: Western Michigan University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-11-01 至 2016-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1120624&HistoricalAwards=false
• 关键词: Ghosts; Evolution; Past; Resurrecting; Extinct

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.

智力优势：蛋白质是分子机器，执行细胞、组织和器官的工作，并最终协调个体的发育。 尽管蛋白质在生命生物学中发挥着重要作用，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，以了解功能如何随时间变化，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灭绝的蛋白质，研究蛋白质功能进化的历史。 在这项研究中，一个3亿年前的祖先蛋白质及其后代将被合成为一组植物代谢产物产生酶，以测试几个假设。第一个要检验的假设是，祖先的酶开始是多面手，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，这些路径允许不同的祖先蛋白进化出相似的功能。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括。 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：这项研究将深入了解植物防御和重要的二级化合物如咖啡因的生产，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产生影响，因为祖先酶提供了有用的点，从那里开始选择所需的活动，因为它们的广谱能力。 本科生和研究生将进行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，并作为手稿详细说明他们的发现。 此外，这里提出的概念性问题是作为一个主题，在高级分子系统发生学和进化过程中，蛋白质进化的适应性假设提出和统计测试的目的是测试的预测。 进化的主题也形成了同伴分子生物学实验室课程的基础，在该课程中，实验用于通过进行基因克隆，蛋白质表达，酶测定和定点诱变来测试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性和概念性的观点，将他们在化学和生物学方面的培训结合起来。