Collaborative Research: Structural, Functional and Evolutionary Basis for the Utilization of a Quinone Methide-Like Mechanism in the Biosynthesis of Plant Specialized Metabolites

合作研究：在植物特化代谢物生物合成中利用醌甲基化物样机制的结构、功能和进化基础

• 批准号: 0718152
• 负责人: Eran Pichersky
• 金额: $ 60.66万
• 依托单位: Regents of the University of Michigan - Ann Arbor
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-15 至 2013-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0718152&HistoricalAwards=false
• 关键词: Collaborative; Research; Structural; Functional; Evolutionary

Plants synthesize a vast array of compounds that facilitate interactions with their environment, from attracting pollinators and seed dispersers to protecting themselves from pathogens, parasites and herbivores. Each plant species has evolved the ability to synthesize a unique set of such chemicals, often classified as secondary or specialized metabolites. While large numbers of such compounds have been identified, our understanding of the enzymes responsible for their biosynthesis is lagging far behind. Of particular interest is a group of metabolites called the phenylpropenes, consisting of a complex set of bioactive volatile chemicals including eugenol and isoeugenol, that have played important roles in human history and continue to serve important agricultural and dietary needs of humankind. The biosynthesis of the phenylpropenes, which are found only in plants, occurs through an unusual reduction reaction. The research groups led by the PI and CoPI have recently identified two representative enzymes responsible for phenylpropene biosynthesis, eugenol and isoeugenol synthases (EGS and IGS), that catalyze the formation of eugenol and isoeuegnol, respectively, from biosynthetic precursors of the plant polymer lignin. The goals of this project are to study the reaction mechanism of the EGS-IGS type enzymes, to identify related enzymes that synthesize other agriculturally and nutritionally important phenylpropenes, and to use this knowledge to modify such enzymes by rational design to create more efficient biosynthetic pathways for economically reliable sources of existing and new high-value phenylpropenes. It is hypothesized that these enzymes use a 2-step mechanism involving a quinone methide-like intermediate that has not previously been studied in detail and that may also be involved in the syntheses of other important, non-phenylpropene specialized compounds. The investigators use a multidisciplinary approach that includes biochemical, genomic and metabolomic approaches to identify new genes and enzymes for phenylpropene biosynthesis, crystallographic studies to solve the 3-dimensional structure of the proteins, and chemical synthesis of substrates and substrate analogs combined with experimentally modified enzymes to examine reaction mechanism. The project also integrates the training of high school students, teachers, undergraduate students and PhD level scientists in state-of-the-art multidisciplinary research.The research will lead to the identification of a number of new enzymes capable of synthesizing phenylpropenes with important agricultural and nutritional properties, and result in a better understanding of how these enzymes work. It will therefore also lead to the rational engineering of new enzymes that can synthesize novel phenylpropenes with enhanced bioactivity as well as the ability to biosynthesize existing compounds more efficiently. Furthermore, since other enzymes that are involved in the synthesis of distinct plant compounds may utilize a similar mechanism, the biochemical research will increase our understanding of the function of these important enzymes as well. The project will also contribute to the multidisciplinary training of the next generation of scientists--including women and minorities--at all levels, as post-docs preparing to assume faculty positions, as graduate students, and as undergraduates preparing to choose a specific career in science. Furthermore, an outreach program to high-school teachers and students will positively influence high school students considering a career in science in the first place. To disseminate the knowledge gained in this project, the research results will be presented at scientific meetings and published in refereed scientific journals, and in addition, the researchers will give occasional talks to the general public about their work and its implications as well as contribute materials to the general public's media such as print, radio and TV as they have done in the past.

植物合成大量的化合物，促进与环境的相互作用，从吸引传粉者和种子传播者到保护自己免受病原体，寄生虫和食草动物的侵害。每种植物都进化出了合成一套独特的此类化学物质的能力，这些化学物质通常被归类为次级或专门代谢物。虽然已经鉴定了大量的此类化合物，但我们对负责其生物合成的酶的理解远远落后。特别令人感兴趣的是一组称为苯丙烯的代谢物，由一组复杂的生物活性挥发性化学物质组成，包括丁香酚和异丁香酚，它们在人类历史上发挥了重要作用，并继续满足人类重要的农业和饮食需求。苯丙烯的生物合成是通过一种不寻常的还原反应进行的，它只存在于植物中。由PI和CoPI领导的研究小组最近确定了两种负责苯丙烯生物合成的代表性酶，丁香酚和异丁香酚脱氢酶（EGS和IGS），它们分别催化植物聚合物木质素的生物合成前体形成丁香酚和异丁香酚。本项目的目标是研究EGS-IGS型酶的反应机理，鉴定合成其他农业和营养重要的苯丙烯的相关酶，并利用这些知识通过合理设计来修饰这些酶，以创造更有效的生物合成途径，为现有和新的高价值苯丙烯提供经济可靠的来源。据推测，这些酶使用2步机制，涉及醌甲基化物样中间体，以前没有详细研究，也可能参与其他重要的，非苯丙烯专门化合物的合成。研究人员使用多学科方法，包括生物化学，基因组学和代谢组学方法来识别苯丙烯生物合成的新基因和酶，晶体学研究来解决蛋白质的三维结构，以及底物和底物类似物的化学合成与实验修饰的酶相结合来检查反应机制。该项目还整合了对高中生、教师、本科生和博士水平科学家的培训，使他们能够进行最先进的多学科研究，该研究将导致鉴定出许多能够合成具有重要农业和营养特性的苯丙烯的新酶，并更好地了解这些酶的工作原理。因此，它也将导致新的酶的合理工程，可以合成具有增强的生物活性以及更有效地生物合成现有化合物的能力的新的苯基丙烯。此外，由于参与不同植物化合物合成的其他酶可能利用类似的机制，生物化学研究也将增加我们对这些重要酶功能的理解。该项目还将促进对下一代科学家-包括妇女和少数民族-进行各级多学科培训，包括准备担任教师职务的博士后、研究生和准备选择科学专业的本科生。此外，对高中教师和学生的推广计划将积极影响高中学生考虑在科学的职业摆在首位。为了传播在这个项目中获得的知识，研究成果将在科学会议上提出，并在经评审的科学期刊上发表，此外，研究人员将不定期地向公众介绍他们的工作及其影响，并像过去一样向公众媒体，如印刷、广播和电视提供材料。