RESTRICTED SCOPE for Participant Support: Glucan phosphatases: a key to designer starches and plant energy storage

Intellectual Merit. As the major energy cache in plants and algae, starch is a central component of human and animal food and a key constituent in many manufacturing processes. Additionally, starch is both a first-generation biofuel and it is vital to future efforts focused on microalgal hydrogen and oil production. Growing starch demand has impacted the drastic rise in corn prices from $85/metric ton in 2002 to $258 in 2012. Therefore, elucidation of pathways controlling starch metabolism is needed in order to develop novel strategies that manipulate them and satisfy the growing starch demand. A key pathway regulating starch metabolism - and one that is required for starch degradation - is reversible phosphorylation of glucose residues in starch outer glucans, rendering the granule surface accessible to glucan hydrolyzing enzymes. Two glucan kinases, identified 7 and 12 years ago, solubilize starch outer glucan chains by phosphorylating different positions on glucose units. Two glucan phosphatases, identified one and five years ago by the PI, exhibit distinct specificities in removing phosphate groups to allow processive glucan hydrolysis. Moreover, the glucan phosphatases do not contain the same domains and preliminary data demonstrate that they posses unique substrate binding mechanisms. Plants lacking the glucan phosphatases exhibit: excess amounts of starch, impaired growth, starch with increased phosphorylation, and accumulation of starch breakdown intermediates. While much progress has been made concerning the biology of reversible starch phosphorylation little is known about the molecular mechanisms regulating glucan phosphatase function. The work in this proposal addresses critical information gaps of this essential pathway. The objective of this CAREER project is to determine the molecular mechanisms of glucan phosphatases with a long-term goal of utilizing engineered glucan phosphatases in planta to increase starch production and generate designer starches, i.e. starches with different biophysical properties. This CAREER proposal will define the function, dynamics, structures, and regulation of glucan phosphatases as well as generate and evaluate engineered glucan phosphatases. Objective I defines the molecular enzymology of glucan phosphatases by determining: the kinetics of substrate specificity, the contribution of each domain to activity, and the role of a unique motif within the phosphatase domain. These results will provide a critical foundational characterization of this enzyme family to guide current and future molecular and genetic engineering. Objective II delineates phosphatase glucan-binding sites and the dynamic structural changes that accompany glucan-binding using deuterium exchange mass spectrometry (DXMS). This research will provide key insights regarding the molecular basis of glucan phosphatase function and will serve as a bridge for additional molecular engineering. Objective III elucidates the structural basis underlying the biological function of glucan phosphatases by determining ligandbound and ligand-free structures, providing the first structures of this enzyme family complexed to its substrate. Cumulatively, these studies will provide a comprehensive profile of how substrate specificity is determined as well as how glucans influence enzyme activity, providing the needed insights for current and future biotechnological exploitation of these enzymes. Broader Impacts. Increasing demand has led to competition for starch among food, biofuels, and industrial manufacturers. An additional concern is that starch processing utilizes hazardous chemicals to modify it for industrial application. Therefore, innovative strategies are needed to increase starch production and to modify starch biophysical properties using less hazardous methods. The PI has proposed integrated and synergistic career goals that will impact our future ability to engineer increased starch quantities and generate designer starches. This project involves multidisciplinary training at several levels. 1) Graduate students will receive national (Kentucky and California) and international (Switzerland) training in laboratory skills, scientific ethics, protein purification, enzyme kinetics, crystallography, DXMS methods, and in data analysis and presentation. Additionally, they will be mentored in career options. 2) Undergraduate students, including NSF REU students and those from the under-represented Appalachia region, will be trained in basic laboratory skills and in the techniques employed in the lab as well as receiving mentorship in career options. 3) The PI will enhance formal undergraduate courses by integrating his research on starch metabolism into a mid-level and upper level undergraduate course. 4) The importance of basic research on the societal and environmental issues of food production, biofuels, and global warming will be presented to high school and undergraduate students as well as the general public at state government events. In addition, these innovative results will be published in peer-reviewed journals and presented at local, regional, national, and international scientific interdisciplinary meetings.