Two large and important questions in plant metabolism are: what are the mechanisms that lead to the large array of compounds produced by plants and how do these mechanisms evolve? Ginger and turmeric contain a large array of compounds that are similar in structure and that appear to be produced by the same biochemical pathway. These compounds, including curcumin, other diarylheptanoids, gingerols and gingerol-like compounds, are produced by polyketide synthase-like enzymes (PKSs). Because these two species produce such a large array of related compounds, they are an ideal system to use to study the molecular and structural basis for differences in production of such compounds in plants. These compounds were chosen as the focus of this project because of their importance to plant defense, to plants' adaptation to the environment, and to human health, and because a large set of genes (41 in total so far) have been identified in ginger and turmeric related to but distinct from known PKSs (such as chalcone synthase) that are expressed differentially between tissues and developmental stages of the two species. This project will focus on identifying which specific members of this gene family are responsible for production of specific curcuminoids, gingerols, other diarylheptanoids or gingerol-related compounds. It will also determine the biochemical function of individual PKSs in ginger or turmeric. This project will then determine the structural basis for differential activity among the different PKSs. This information will address the important questions of how changes in enzyme structure lead to differences in enzyme function and how these changes evolve in the context of plant metabolism.
Broader Impacts This project will impact understanding of the evolution of enzyme function, especially with regard to how enzymes develop new functions. This in turn will impact understanding of factors that lead to compound diversification in the plant kingdom and ultimately to plant diversity in the biosphere. This project will also produce information that could be used to better tailor the production of specific compounds in target plants, increasing the ability to produce "designer plants". Undergraduate students and high school teachers will work with post-doctoral fellows in this research. The high school teachers (supported by summer internships) will take purified PKS proteins to their classrooms and then will work with their students to crystallize these proteins. These proteins will include altered forms of the enzymes, which will be produced to probe the questions of structure and function in this enzyme class, as well as control proteins that easily crystallize, so that the students will have a successful experience. Thus, the high school teachers and their students will work directly on a research project that will have potentially important findings regarding enzyme function, and they will learn about structural biology in the process.
Ginger and turmeric are known worldwide for their medicinal and culinary importance. Many of the beneficial properties of these plants are due to two classes of related compounds that they produce, known as the curcuminoids and gingerols, and especially to the presence of curcumin in turmeric and tetrahydrocurcumin in ginger. This project sought to further our understanding of how these compounds are produced in these plants, how the metabolic network that produces them is organized and how it evolved in these plants. Several classes of enzymes were identified as playing important roles in controlling the biosynthesis of these compounds, including polyketide synthases, double bond reductases and thioesterases. A genomics-based approach identified many genes representing these enzyme classes in the tissues that produced the curcuminoids, particularly in the rhizome, or underground stem of these plants, which is the tissue from ginger and turmeric that is used as a spice. Several different polyketide synthases were identified as belonging to two subclasses, which work together to produce the backbone structure of the curcuminoids. Members of these subclasses were found to have fairly strict substrate specificities and were expressed in different tissues in the plants, which explains how different subclasses of these compounds can be differentially produced in different tissues. A similar situation was found for the double bond reductases, which were found to act on the formed curcuminoid background, and not on an upstream intermediate in the pathway to these compounds. Again, different double bond reductases with different substrate specificities were found to be expressed in different tissues, such as leaves or rhizomes. In addition to these genes, specific thioesterase activities were identified in turmeric and ginger tissues that act differentially on the precursors to the curcuminoids, on the hydroxycinnamoyl-CoA esters. The thioesterase enzymes may also play roles in determining the final distribution of curcuminoid sub-classes in the tissues of these plants. Thus, this project identified many enzymes involved in production of the curcuminoids, including curcumin and tetrahydrocurcumin, which are anti-inflammatory compounds and show great promise in cancer chemoprevention and treatment. The large diversity of these compounds found in these plants can be explained by radiation of specific gene families, leading to a multitude of similar enzymes that have similar but distinct activities, and that therefore can be used by the plant to modulate or control production of specific compounds within its tissues. Finally, some of these enzymes (particularly the double bond reductases) have been purified and crystallized, allowing for generation of structural models of the proteins, which will further enable detailed characterization of how these enzymes function, how they evolved, and how their activities control the generation of a large array of related compounds in these plants.
