Structural Basis for Reversible Glucan Phosphorylation by Glucan Dikinases and Glucan Phosphatases

Plants store glucose in the form of starch, an insoluble macromolecule that is synthesized and degraded diurnally. Glucan phosphatases play a critical role in starch breakdown, working with glucan dikinases in a cyclic phosphorylation and dephosphorylation. Herein, we present a novel glucan phosphatase Like SEX Four3 (LSF3) from Physcomitrella patens. Structural and enzymatic studies demonstrate that LSF3 is a dimeric glucan phosphatase that displays robust specific glucan phosphatase activity. Dimerization is shown to be essential for LSF3 stability and function. Structure-guided bioinformatic analysis shows that LSF3 is found in early land plants and green algae. Building upon our understanding of the mechanism of glucan phosphatase activity, we engineered the active site of a protein tyrosine phosphatase from a thermophilic bacterium. Additionally, two tandem carbohydrate binding domains were fused to the N-terminus of the mutant protein. We show that the chimeric protein is able to release phosphate from starch and synergize with amylase, enhancing maltose release at elevated temperatures. The reversible glucan phosphorylation system can also be found in non-photosynthetic organisms. The genome of Toxoplasma gondii, an obligate parasite infecting one-third of world population, encodes a glucan phosphatase (TgLaforin) and a glucan water dikinase (TgGWD). Herein, we show that TgLaforin possesses a unique split-CBM20 with long disordered regions. Structural modeling and HDX data show that TgLaforin exists as a dimer mediated exclusively through the phosphatase domain. Additionally, a crystal structure of TgGWD was determined at 2.7 Å. Structural analysis, structure modeling, and mutagenesis studies suggest that TgGWD utilizes a swiveling mechanism to transfer phosphate from ATP to glucan substrates. Our studies provide valuable insights into the mechanism of reversible glucan phosphorylation and lay the groundwork for developing new therapeutics to target carbohydrate utilization in T. gondii