Glucan phosphatases are a newly defined class of enzymes that dephosphorylate the complex carbohydrate substrate starch and glycogen, thereby regulating their metabolism. Plants and algae contain two glucan phosphatases called Starch EXcess 4 (SEX4) and Like Sex Four 2 (LSF2) that dephosphorylate starch and are essential for its diurnal breakdown in plant leaves. Vertebrates and a few unicellular eukaryotes contain one glucan phosphatase called Laforin that dephosphorylates glycogen, thereby promoting glycogen solubility and hydrolytic enzyme accessibility. Glucan phosphatases are members of the Dual Specificity Phosphatase (DSP) family of enzymes. Previous studies have determined the structural basis for interaction between DSPs and protein, lipid, and nucleic acid substrates, but the structural basis of phosphatase-glucan interaction was previously unknown. We determined the structure of LSF2 with and without a maltohexaose-phosphate substrate bound to a resolution of 2.3� and 1.65�, respectively. We determined that LSF2 binds maltohexaose-phosphate via an aromatic channel within an extended phosphatase active site. The LSF2 aromatic channel positions the glucan chain in a C3- orientation, thus revealing the structural basis for previously determined LSF2 C3- phospho-glucan substrate specificity. We also determined that LSF2 undergoes a substrate-dependent rearrangement that influences the catalytic state of LSF2. Lastly, we determined that LSF2 binds starch via two non-catalytic glucan binding sites that are necessary for starch binding and dephosphorylation. The combination of non-catalytic glucan binding sites with a DSP site in LSF2 represents a convergence of structural motifs found in protein phosphatases and amylases. These data provide useful insights into the mechanism of glucan phosphatase activity and provide a basis for additional engineering of these agriculturally relevant enzymes.

Structure of starch phosphatase Like Sex Four2 (LSF2) � convergent mechanisms of glucan interaction and dephosphorylation

246th National Meeting (2013)

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