Biophysical and Structural Characterization of Glucan Phosphatase SEX4 from Zea mays.

Plants store energy in starch, a glucan made of two polymers of glucose- amylose and amylopectin. Phosphorylation, the only known in vivo modification of starch, controls the structure, function, and physicochemical properties of the starch granule and thereby modulates the starch degradation at night. Reversible starch phosphorylation has been increasingly appreciated as a key regulatory step controlling diurnal starch metabolism. Glucan phosphatase Starch Excess4 (SEX4) is an allosteric enzyme that binds to and dephosphorylates starch at the C6 and C3 positions of glucose residues. Plants lacking glucan phosphatases exhibit excess starch and starch with increased phosphorylation. However, our understanding of how SEX4 binds to the semicrystalline starch stricture and regulates the dephosphorylation mechanism is incomplete. Previously, the x-ray crystal structure of catalytically inactive Arabidopsis thaliana SEX4 bound to maltohexaose has been described. Here we present the x-ray crystal structure of wild-type SEX4 from Zea mays to a resolution of 2.9 A. Utilizing hydrogen-deuterium exchange spectroscopy and differential scanning fluorometry, we intend to understand how SEX4 interacts with its glucan substrate for an efficient starch dephosphorylation mechanism. We also investigated the ability of SEX4 to enhance in-vitro starch degradation. Our findings reveal that SEX4 from cereal crops such as Zea mays has the increased ability to enhance starch degradation than the SEX4 orthologs from storage crops.