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

植物合成大量化合物，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。每一种植物都进化出了合成一套独特的化学物质的能力，这些化学物质通常被归类为次级或专门的代谢物。虽然已经发现了大量这样的化合物，但我们对负责其生物合成的酶的了解远远落后。特别令人感兴趣的是一组被称为苯丙烯的代谢物，它由一系列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。只存在于植物中的苯丙烯的生物合成是通过一种不寻常的还原反应进行的。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，丁香酚和异丁香酚合成酶（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。该项目的目标是研究EGS-IGS型酶的反应机理，识别合成其他具有重要农业和营养价值的苯丙烯的相关酶，并利用这些知识通过合理设计对这些酶进行修饰，为经济可靠的现有和新的高价值苯丙烯来源创造更有效的生物合成途径。据推测，这些酶使用两步机制，涉及一种醌类中间体，这种中间体以前没有被详细研究过，也可能涉及其他重要的非苯基丙烯特异性化合物的合成。研究人员采用多学科方法，包括生物化学、基因组学和代谢组学方法来鉴定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，化学合成底物和底物类似物结合实验修饰酶来研究反应机制。该项目还整合了对高中生、教师、本科生和博士级科学家进行最先进的多学科研究的培训。这项研究将导致鉴定出一些能够合成具有重要农业和营养特性的苯丙烯的新酶，并使人们更好地了解这些酶是如何工作的。因此，这也将导致新的酶的合理工程，可以合成具有增强生物活性的新型苯丙烯，以及更有效地生物合成现有化合物的能力。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，生化研究也将增加我们对这些重要酶功能的了解。该项目还将促进对包括女性和少数族裔在内的各级下一代科学家的多学科培训，包括准备担任教职的博士后、准备选择特定科学职业的研究生和准备选择特定科学职业的本科生。此外，针对高中教师和学生的外展计划将对首先考虑从事科学职业的高中生产生积极影响。为了传播在这个项目中获得的知识，研究结果将在科学会议上展示，并发表在经过评审的科学期刊上，此外，研究人员将不定期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。

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.