Starch is a water-insoluble glucose biopolymer used as an energy cache in plants and is synthesized and degraded in a diurnal cycle. Reversible phosphorylation of starch granules regulates the solubility and, consequentially, the bioavailability of starch glucans to degradative enzymes. Glucan phosphatases release phosphate from starch glucans and their activity is essential to the proper diurnal metabolism of starch. Previously, the structural basis of glucan phosphatase activity was entirely unknown. The work in this dissertation outlines the structural mechanism of activity of two plant glucan phosphatases called Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2). The crystal structures of SEX4 and LSF2 were determined with and without phosphoglucan ligands bound, revealing the basis of their interaction with an endogenous substrate. The data show that SEX4 and LSF2 interact with starch glucans via distinctive mechanisms. SEX4 binds glucan chains via an aromatic-rich pocket spanning its Carbohydrate Binding Module (CBM) and catalytic Dual Specificity Phosphatase (DSP) domains. Conversely, LSF2 lacks a CBM and, instead, binds glucans at two non-catalytic surface-binding sites that are located distally from its active site. In addition, it was previously reported that SEX4 and LSF2 act upon distinct phospho-glucan substrates: SEX4 preferentially dephosphorylates the C6-position of starch glucans and LSF2 exclusively dephosphorylates the C3- position. The data herein reveal that SEX4 and LSF2 contain differences in their active site topology that serve to position the glucan chain in opposite orientations, therefore accounting for the differences in substrate specificity. Using these insights, SEX4 was engineered with reversed substrate specificity, i.e. preferential C3-specific activity. Previous work has established the interaction between phosphatases and protein, lipid, and nucleic acids; however, the current study represents the first insights into phosphatase interaction with carbohydrate substrates. In addition, the insights gained provide a model that will be used in future studies with the mammalian glucan phosphatase laforin, which is linked to neurodegeneration and the fatal epileptic disorder Laforaʼs Disease.

Lafora disease (LD) is a rare yet invariably fatal form of epilepsy characterized by progressive degeneration of the central nervous and motor systems and accumulation of insoluble glucans within cells. LD results from mutation of either the phosphatase laforin, an enzyme that dephosphorylates cellular glycogen, or the E3 ubiquitin ligase malin, the binding partner of laforin. Currently, there are no therapeutic options for LD, or reported methods by which the specific activity of glucan phosphatases such as laforin can be easily measured. To facilitate our translational studies, we developed an assay with which the glucan phosphatase activity of laforin as well as emerging members of the glucan phosphatase family can be characterized. We then adapted this assay for the detection of endogenous laforin activity from human and mouse tissue. This laforin bioassay will prove useful in the detection of functional laforin in LD patient tissue following the application of therapies to LD patients. We subsequently developed an in vitro readthrough reporter system in order to assess the efficacy of aminoglycosides in the readthrough of laforin and malin nonsense mutations. We found that although several laforin and malin nonsense mutations exhibited significant drug-induced readthrough, the location of the epitope tag used to detect readthrough products dramatically affected our readthrough results. Cell lines established from LD patients with nonsense mutations are thus required to accurately assess the efficacy of aminoglycosides as a therapeutic option for LD. Using hydrogen-deuterium exchange mass spectrometry (DXMS), we then gained insight into the molecular etiology of several point mutations in laforin that cause LD. We identified a novel motif in the phosphatase domain of laforin that shares homology with glycosyl hydrolases (GH) and appears to play a role in the interaction of laforin with glucans. We studied the impact of the Y294N and P301L LD mutations within this GH motif on glucan binding. Surprisingly, these mutations did not reduce glucan binding as expected, rather enhancing the binding of laforin to glucans. These findings elucidate the mechanism by which laforin interacts with and acts upon glucan substrates, providing a target for the development of therapeutic compounds.