Molecular Definition of the Unique Phenylpropanoid Radical Coupling Mechanisms of Dirigent Proteins, Their Homologues, and Associated Metabolism: A Discovery-Based Approach

对植物中氧化自由基偶联/聚合化学的控制是这些生物获得陆基栖息地的决定性适应。在这样做的过程中，这些不同的植物形式精心设计了新的生物化学方法，以产生大量具有重要作用的苯丙烷类(醋酸酯)天然产物，例如在植物防御中以及后来在人类健康中的应用(包括作为抗病毒、抗癌和杀菌剂)。这些代谢途径还产生了结构复杂的生物聚合物，木质素，它在很大程度上决定了木质结构特性，以及木素(软木组织中的)，使植物能够在干燥的环境中生存。这与其他生物体类型(如哺乳动物)不同，在其他生物体类型中，自由基化学没有得到很好的控制，例如在人类中，这种缺乏控制可能导致更快的衰老和对各种疾病的易感性。这个项目将调查植物中这种自由基偶联/聚合化学是如何实际控制的，它们是如何进化的，以及建立在发现介导性蛋白质的基础上。后者是蛋白质控制自由基-自由基偶联的第一个例子，因此，将要进行的研究将确定参与自由基结合、稳定和最终其产物形成的蛋白质的关键特征的性质。这项研究中将使用的技术是电子顺磁共振光谱、X射线结晶学和定点突变，即能够识别控制这种自由基化学的直接蛋白质的关键特征以及相关的多酚氧化酶和区域特异性偶联酶的关键特征。这反过来将使我们有可能确定大自然实际上是如何制造如此多样和复杂的天然产品的；这可以预期在许多不同的事业中具有巨大的价值，例如导致新材料的受控自由基聚合过程、纳米技术、医学和蛋白质进化的研究。广泛影响NSF的更广泛的影响是鼓励和培养本科生、研究生、博士后、访问科学家和高中教师级别的年轻科学家(特别是来自代表不足和服务不足的群体)发展他们的职业生涯。后一种机制提供了一个极好的途径，将最新的技术见解和进步引入学校。除了上述科学益处，这些研究人员还参与了许多其他外展活动，包括华盛顿州立大学(WSU)植物生物化学暑期课程、华盛顿州立大学一年一度的核磁共振研讨会；以及针对高中生和普通公众的一般性讲座，例如关于转基因食品的利弊和饮食中植物化学物质的多样性的讲座。

The control of oxidative free radical coupling/polymerization chemistry in plants was a decisive adaptation of such organisms in order to access a land-based habitat. In so doing, these diverse plant forms elaborated new biochemistries to generate a plethora of phenylpropanoid (acetate) natural products with important roles, e.g. in plant defense and later with applications in human health (including as antiviral, anticancer and bactericidal agents). These metabolic pathways also gave the structurally complex biopolymers, the lignins, which largely determine woody textural properties, and the suberins (in cork tissue) that enabled plants to survive in desiccating environments. This contrasts with other organismal types (e.g. with mammals), where radical chemistry is not as well controlled, e.g. in humans, where this lack of control can lead to more rapid aging and increased susceptibility to various diseases.This project will investigate the basis of how such radical coupling/polymerization chemistries are actually controlled in plants, and how they evolved, as well as building upon discovery of dirigent proteins. The latter represents the first example of a protein controlling radical-radical coupling, and thus the research to be undertaken will determine the nature of the key features on the protein involved in radical binding, stabilization and ultimately its product formation. The technologies that will be used in this study are those of electron paramagnetic resonance spectroscopy, X-ray crystallography and site-directed mutagenesis, i.e. procedures, which will enable identification of the key features in dirigent proteins that control such radical chemistries, as well as those in related polyphenol oxidase and regiospecific coupling enzymes. This, in turn, will make it possible to establish how nature actually makes such diverse and complex natural products; this can be anticipated to be of immense value in many different undertakings, such as controlled radical polymerization processes leading to new materials, in nanotechnology, in medicine, and in the study of protein evolution.Broader ImpactThe broader impact of NSF support is in the ability to encourage and nurture young scientists (particularly from underrepresented and underserved groups) at the undergraduate, graduate, postdoctoral, visiting scientist and high school teacher levels in developing their careers. The latter mechanism gives an excellent avenue for introducing the latest technological insights and advances into schools. In addition to the scientific benefits described above, these investigators are involved in a number of other outreach activities including the Washington State University (WSU) Plant Biochemistry Summer Course, the annual WSU Nuclear Magnetic Resonance Workshop; and general talks to high school students and the general public, for example on the pros and cons of genetically modified foods and the diversity of dietary phytochemicals.