Insights into the mechanism of polysaccharide dephosphorylation by a glucan phosphatase.

M embers of the protein tyrosine phosphatase (PTP) family usually catalyze the dephosphorylation of protein substrates. However, some enzymes from a subclass of the PTPs, the dual specificity phosphatases (DSPs), dephosphorylate nonproteinaceous substrates such as lipids and polysaccharides (1–3). In PNAS, Vander Kooi et al. (4) describe the crystal structure of the glucan phosphatase, starch excess 4 (SEX4), providing the first insight into the structural basis of complex carbohydrate dephosphorylation by this emerging family of phosphatases. Glycogen and amylopectin, the main constituent of starch, are branched polymers of glucose that share a basic purpose—both serve as osmotically neutral glucose stores, synthesized under conditions of nutritional plenty for utilization in times of metabolic need. As examples, liver glycogen is synthesized in the fed state and then mobilized to maintain blood glucose levels during fasting (5); leaves of green plants synthesize starch in the daytime by using energy from photosynthesis that is then used to generate maltose and glucose for energy production during darkness (6). They share a fundamental chemical structure—both contain glucose units connected by α-1,4-glycosidic linkages with branch points formed by α-1,6-glycosidic linkages (Fig. 1A). Lastly, and most relevant to our discussion here, both polysaccharides contain small amounts of covalently linked phosphate, one phosphate per 500–1,500 glucoses in glycogen (3, 7) and one per 150–300 glucoses in amylopectin (8). Recent research has focused on the covalent phosphate in the two polysaccharides. In amylopectin, the phosphate is present as C3 and C6 phosphomonoesters and is introduced by the concerted action of two dikinase enzymes, glucan water dikinase (GWD) and phosphoglucan water dikinase (PWD) (9) (Fig. 1D). Phosphate is released by SEX4, a member of the dual specificity protein phosphatase family that additionally contains a C-terminal carbohydrate binding module (CBM) of the CBM48 subtype (Fig. 1B). Much less is known about the metabolism of the covalent phosphate in glycogen. Animals lack dikinases equivalent to GWD and PWD, and the origin of the phosphate is only now being worked out. Also, the chemical nature of the phosphate linkage to glycogen is uncertain. However, its hydrolysis is catalyzed by a DSP, called laforin (3), that has important similarities to SEX4. The laforin DSP domain is also flanked by a CBM, in this case an N-terminal CBM20 subtype (Fig. 1B). Laforin is primarily restricted to vertebrates, although it is found in certain protists that produce a scarcely branched glucose polymer called floridean starch (10). SEX4 and laforin may represent an example of convergent evolution (11) and, indeed, laforin can partially rescue the phenotype of plants defective in SEX4 (10). What is the function of the phosphate in amylopectin and glycogen? The best evidence to date comes from genetics. SEX4 is named for the phenotype resulting from its mutation, starch excess, and is involved in the catabolism of starch (12). The branching in amylopectin is discontinuous, leading to clusters of aligned polyglucose helices that form insoluble crystalline lamellae that are metabolically inert (Fig. 1C) (13). The prevailing view is that phosphorylation of glucose residues in these crystalline regions by the GWD and PWD dikinases disrupts the structure to allow hydration, thereby allowing access to degradative enzymes, β-amylases (BAM1 and BAM3) and isoamylase (ISA3), that generate oligosaccharides (12) (Fig. 1D). In the absence of SEX4 to hydrolyze the C3 and C6 monoesters, starch degradation is impaired and phospho-oligosaccharides accumulate (12). Thus, the function of amylopectin phosphorylation is in a transient step of normal starch degradation. CBM20 CBM48 cTP