Structural Basis for Glucan Phosphatase Function in Photosynthetic Organisms

Overview Photosynthetic organisms store energy in the form of the carbohydrate polymer starch. Starch is water-insoluble and as such, it is ideally suited as an energy storage molecule. However, catabolic enzymes, including the amylases that break down starch, are unable to effectively access this insoluble energy cache. Recently, the critical importance of reversible phosphorylation of starch glucans has been shown to play a critical role in plant metabolism. Phosphorylation of the outer glucose residues of starch by glucan dikinases renders the granule surface accessible to starch degrading amylases, while removal of the phosphate groups by glucan phosphatases allows processive glucan hydrolysis. Thus, starch degradation proceeds by a cyclic process of phosphorylation, degradation, and dephosphorylation. The key biological role of reversible phosphorylation has been increasingly appreciated since plants lacking the glucan phosphatases exhibit excess amounts of starch, impaired growth, and starch with increased phosphorylation. While of clear biological importance, little is known about the molecular mechanisms of reversible starch phosphorylation. We will define the structural basis for the specificity and functional coupling of the members of the glucan phosphatase family, elucidating the critical function of this modification. Taken together, these studies will define the structural mechanism of glucan phosphatases in diverse photosynthetic organisms and harness this unique chemical carbohydrate modification. Intellectual Merit. This proposal focuses on four objectives to determine the molecular mechanisms of glucan phosphatases across the kingdoms of photosynthetic organisms and develop methods to harness their unique activity. Objective I will define the specificity of the plant glucan phosphatases Starch Excess-Four (SEX4), focusing on specific glucan engagement and activity. Objective II will elucidate the enigmatic function of the Like-SEX4-1 (LSF1) protein. LSF1 has a clear role in starch metabolism, but the molecular function for this activity is unclear. Objective III will characterize the novel glucan phosphatase from the single-cell, red alga Cyanidioschyzon merolae. Coupled structural and functional studies will define the function of the single glucan phosphatase from an extremophile, and compare its structure and activity to plant and vertebrate glucan phosphatases. Finally, Objective IV will define the enzymatic coupling of glucan phosphatases with other catabolic enzymes, and harness their activity in starch utilization. This will inform on the biological role and coupling of glucan phosphatases and open up new avenues for their utilization. In addition to its critical role in plants and algae, starch is a key component in many aspects of daily life, including nutrition, biofuel production, and industrial processing. Understanding and controlling glucan phosphorylation has considerable promise as an innovative strategy to both understand the chemistry of this process in living systems and harness it to engineer and utilize starch. Broader Impacts. Glucan phosphorylation has been shown to play a key biological role and has the potential for significant utility in starch utilization. Both fundamental scientific insight and innovative technologies are needed. These innovative approaches involve not only fundamental and applied research, but also prepare the next generation of highly skilled young scientists with training in cutting edge interdisciplinary approaches. This project involves training and development at multiple levels. Graduate students will receive training in interdisciplinary laboratory skills, data analysis, presentation, and scientific ethics. Undergraduate students, including NSF REU students and those from the under-represented Appalachia region, will be trained in cutting-edge laboratory skills. Students at all levels will be jointly mentored in career options. Our research on plant enzymes important for starch metabolism will be incorporated into the undergraduate Fundamentals of Biochemistry course. Results from this work will be will be published in peer-reviewed journals and presented at regional and national scientific meetings. Additionally, we will present this research to local and regional Primarily Undergraduate Institutions, emphasizing the importance of basic research on societal and environmental issues.