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

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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.