Structural Studies of a Novel Glucan Phosphatase from the Red Alga Cyanidioschyzon Merolae

Background Glucan phosphatases are a unique subset of the Dual Specificity Phosphatase (DSP) family that binds and dephosphorylates carbohydrate substrates. They are found in widely divergent organisms ranging from extremophilic single‐cell red algae to humans and act on different carbohydrate (starch or glycogen) substrates. Red algae, Cyanidioschyzon merolae ( Cm ) possesses a single glucan phosphatase with three structural domains which are different in family and organization compared to other known glucan phosphatase family members. Further, bioinformatics analysis suggested that it is most similar to a vertebrate glucan phosphatase rather than plant glucan phosphatases. This is intriguing, since its endogenous substrate is starch rather than glycogen. Hypothesis Cm‐laforin is uniquely stable and active glucan phosphatase and can be an important partner with other catabolic enzymes in the process of starch metabolism. Objective Explore the structural basis for Cm‐laforin's function as a novel glucan phosphatase. Methods We expressed and purified the full, tandem, and isolated carbohydrate binding (CBM) and phosphatase (DSP) domains of Cm‐laforin to study their thermal stability, phosphatase function, and domain coupling. We utilized para‐nitrophenylphosphate (p‐NPP) substrate for generic and amylopectin in malachite green assay for specific phosphatase activities determination. Differential scanning fluorimetry (DSF) and starch binding assays were utilized for glucan binding studies. The oligomeric state of laforin was determined by Size Exclusion Chromatography with Multi‐Angle static Light Scattering (SEC‐MALS). X‐ray crystallography was utilized for structural studies, which allowed structure‐guided mutagenesis. Results Cm‐laforin is found to be a stable and highly active glucan phosphatase. The full three‐domain protein was found to be highly active and have maximum phosphatase activity at 55°C. Further, it possesses 2‐fold higher specific glucan phosphatase activity compared to vertebrate laforin orthologs. Utilizing the tools of structural biology, we explored the importance of different amino acids of the DSP domain required for its stability. We determined the structure of the DSP domain at 1.5Å and found that Cm‐laforin utilizes a significant number of unique electrostatic interactions centered on D89 to maintain its significantly higher stability. We also found that CBM1 and DSP domain can be expressed and function in isolation, but CBM1 and linker region between CBM2 and DSP are required for the proper folding and stability of CBM2. We further demonstrate that Cm‐laforin exists as a monomer, and that the tandem CBM domains are required for high affinity glucan binding and enzymatic coupling during starch degradation. Conclusion Cm‐laforin is enzymatically highly active and stable phosphatase with unique structural and functional features that underlie its role as a specific glucan phosphatase. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .