Structural basis of activity for plant glucan phosphatases

Glucan phosphatases (GPs) are a subset of dual‐specificity phosphatases (DSPs) that dephosphorylate plant starch, leading to its efficient degradation. A. thaliana contains three known GPs that have sequence homology to laforin; a GP associated with the neurodegenerative disorder Lafora's disease. The main objective of the study was to deduce what effect specific regions of the protein had on overall GP activity. We used X‐ray crystallography to determine the ligand‐free structure of S tarch EX cess 4 (SEX4), the founding member of the plant GPs. Site‐directed mutagenesis and in vitro phosphatases assays measured the importance of certain residues and regions. Our results demonstrate that the carbohydrate‐binding module (CBM) in SEX4 and L ike S ex F our 1 (LSF1) increases glucan binding. We also demonstrated that residues that have homology to glycosyl hydrolase regions contribute to GP activity, especially in L ike S ex F our 2 (LSF), which lacks a CBM. Certain conserved residues found adjacent to the active site, when mutated, led to an overall decrease in activity. Our structure also revealed a conserved c‐terminal residue that is essential for proper folding of the GPs. We conclude that each of these residues contributes to overall activity and differences in these regions between the three plant GPs explain their relative activity in vitro and will provide a basis to determine their unique roles in the cell. Supported by NIH grants R00NS061803, P20RR020171, and R01NS070899, and University of Kentucky College of Medicine startup funds to M.S.G.