Defining Glucan Dikinase Phosphorylation of Starch from Multiple Kingdoms

Plants store energy in the form of the carbohydrate polymer starch. Starch is ideally suited as an energy storage molecule due to its stability. However it is water insoluble, limiting the accessibility of catabolic enzymes including the amylases that break down starch. Plants generate transitory starch during the day and degrade it at night to utilize the energy cache. Natural starch degradation in planta requires reversible starch phosphorylation of glucose residues in the outer shell. When transitioning from synthesis to degradation, plants utilize glucan dikinases to phosphorylate the outer glucose residues within starch, thereby rendering the granule surface accessible to starch degrading amylases. This solubilizes the outer starch surface permitting degradation of surface glucans by amylases. Glucan phosphatases remove the phosphate groups to allow processive glucan hydrolysis (Figure). Thus, transitory starch degradation proceeds by a cyclic process of phosphorylation, degradation, and dephosphorylation. The key biological role of reversible phosphorylation has been increasingly appreciated. Plants lacking the glucan dikinases exhibit excess amounts of starch, impaired growth, and aberrant starch with decreased phosphorylation. While of clear biological importance, little is known about the structural mechanisms of starch phosphorylation. In addition to its critical role in plants, starch is a key component in many aspects of daily life, including nutrition, biofuel production, and industrial processing: 50-80% of daily caloric intake comes from starch; >20% of corn starch produced in the U.S. is converted into ethanol; and starch is a cheap and renewable industrial feedstock for producing paper, textiles, plastics, and pharmaceuticals. Increasing demand has led to competition for starch among food, biofuels, and industrial manufacturers. Growing starch demand has impacted the drastic rise in corn prices from $85/metric ton in 2002 to $258 in 2012. An additional concern is that starch processing requires hazardous chemicals in order to modify it for industrial application. Starch-based feedstocks are generated by an approach utilizing harsh chemical (acid and base treatment) and physical (>100¢ªC) extremes combined with large quantities of amylase enzymes to degrade or modify native starch for industrial applications. Innovative strategies are needed to provide safer, more efficient, and cheaper starch-based feedstocks and defining the glucan kinases is a key step in unlocking their industrial potential. There are key gaps in our knowledge of how glucan dikinases function and how to harness this natural process for efficient bioprocessing of starch. The work in this proposal addresses each these critical deficiencies. The objective of this project is to determine the molecular mechanisms of glucan dikinases with a three-prong approach focused on: 1) determining their molecular enzymology from algae to higher plants by defining the function of each domain within the proteins, including active site specificity and phosphorylation mechanism; 2) defining the structure/function relationship and dynamics of GWD domains; and 3) elucidating the biological function and localization of the algal glucan dikinase and phosphatase. Objective I defines the molecular enzymology of glucan dikinases by determining: the contribution of each domain to activity and how the active site achieves specificity from algae to plants. These results will provide a critical foundational characterization of this enzyme family to guide current and future molecular engineering. Objective II elucidates the structural basis underlying the biological function of glucan dikinases by determining ligand-bound and ligand-free structures, providing the first structures of this enzyme family. 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. Objective III establishes the cellular function of glucan dikinases and phosphatases by defining their subcellular localization. This Objective will establish a cellular system to probe reversible starch phosphorylation. This proposal will define starch phosphorylation from enzymology to structure and dynamics to cell biology.