Defining the Regulatory Roles of Glucan Phosphatases in Transitory Starch Degradation

Abstract

Plants generate starch during the day and metabolize it at night to store and use for cellular energy. The glucan phosphatase Starch EXcess4 (SEX4) plays a crucial role in transitory starch degradation via its involvement in the reversible starch phosphorylation mechanism. Previous structural studies have shown that SEX4 preferentially binds to and dephosphorylates starch at the C6 position of glucosyl residues, but there remains little data on how SEX4 navigates and interacts with the complex starch granule. Further, enzyme kinetics and regulatory mechanisms of SEX4 are currently not well understood. We employed both generic and substrate specific phosphatase assays to determine kinetics and substrate binding mechanisms of SEX4. Upon use of a generic phosphatase substrate, p‐nitro phenyl phosphate (p‐NPP), SEX4 followed Michaelis‐Menten kinetics. A sigmoidal curve observed for the physiological substrate amylopectin suggests a non‐ Michaelis‐Menten behavior for SEX4 and provides experimental evidence for a possible allosteric regulatory mechanism of SEX4. The enhanced dephosphorylation rates obtained for generic phosphatase assays with SEX4 preincubated with different domains of starch structure strongly suggest a conformational change within the active site of SEX4 upon glucan binding. Collectively, our finding suggests a novel posttranslational regulatory mechanism of SEX4 that is necessary for the regulation of transitory starch degradation at night.