Phosphorylases are key enzymes of carbohydrate metabolism. Structural studies have provided explanations for almost all features of control and substrate recognition of phosphorylase but one question remains unanswered. How does phosphorylase recognize and cleave an oligosaccharide substrate? To answer this question we turned to the Escherichia coli maltodextrin phosphorylase (MalP), a non‐regulatory phosphorylase that shares similar kinetic and catalytic properties with the mammalian glycogen phosphorylase. The crystal structures of three MalP–oligosaccharide complexes are reported: the binary complex of MalP with the natural substrate, maltopentaose (G5); the binary complex with the thio‐oligosaccharide, 4‐ S ‐α‐D‐glucopyranosyl‐4‐thiomaltotetraose (GSG4), both at 2.9 Å resolution; and the 2.1 Å resolution ternary complex of MalP with thio‐oligosaccharide and phosphate (GSG4‐P). The results show a pentasaccharide bound across the catalytic site of MalP with sugars occupying sub‐sites −1 to +4. Binding of GSG4 is identical to the natural pentasaccharide, indicating that the inactive thio compound is a close mimic of the natural substrate. The ternary MalP–GSG4‐P complex shows the phosphate group poised to attack the glycosidic bond and promote phosphorolysis. In all three complexes the pentasaccharide exhibits an altered conformation across sub‐sites −1 and +1, the site of catalysis, from the preferred conformation for α(1–4)‐linked glucosyl polymers.

A glucopyranose spirohydantoin (a pyranose analogue of the potent herbicide, hydantocidin) has been identified as the highest affinity glucose analogue inhibitor of glycogen phosphorylase b (GPb). In order to elucidate the structural features that contribute to the binding, the structures of GPb in the native T state conformation and in complex with glucopyranose spirohydantoin have been determined at 100 K to 2.0 Å and 1.8 Å resolution, respectively, and refined to crystallographic R values of 0.197 (R_free 0.248) and 0.182 (R_free 0.229), respectively. The low temperature structure of GPb is almost identical to that of the previously determined room temperature structure, apart from a decrease in overall atomic temperature factors (〈B〉 room temperature GPb = 34.9 Ų; 〈B〉 100 K GPb = 23.4 Ų). The glucopyranose spirohydantoin inhibitor (Kⁱ = 3.0 μM) binds at the catalytic site and induces small changes in two key regions of the protein: the 280s loop (residues 281‐286) that results in a decrease in mobility of this region, and the 380s loop (residues 377‐385) that undergoes more significant shifts in order to optimize contact to the ligand. The hydantoin group, that is responsible for increasing the affinity of the glucose compound by a factor of 10³, makes only one hydrogen bond to the protein, from one of its NH groups to the main chain oxygen of His377. The other polar groups of the hydantoin group form hydrogen bonds to five water molecules. These waters are involved in extensive networks of hydrogen bonds and appear to be an integral part of the protein structure. Analysis of the water structure at the catalytic site of the native enzyme, shows that five waters are displaced by ligand binding and that there is a significant decrease in mobility of the remaining waters on formation of the GPb‐hydantoin complex. The ability of the inhibitor to exploit existing waters, to displace waters and to recruit new waters appears to be important for the high affinity of the inhibitor.

In animals, glycogen phosphorylase (GP) exists in an inactive (T state) and an active (R state) equilibrium that can be altered by allosteric effectors or covalent modification. In Escherichia coli , the activity of maltodextrin phosphorylase (MalP) is controlled by induction at the level of gene expression, and the enzyme exhibits no regulatory properties. We report the crystal structure of E.coli maltodextrin phosphorylase refined to 2.4 Å resolution. The molecule consists of a dimer with 796 amino acids per monomer, with 46% sequence identity to the mammalian enzyme. The overall structure of MalP shows a similar fold to GP and the catalytic sites are highly conserved. However, the relative orientation of the two subunits in E.coli MalP is different from both the T and R state GP structures, and there are significant changes at the subunit‐subunit interfaces. The sequence changes result in loss of each of the control sites present in rabbit muscle GP. As a result of the changes at the subunit interface, the 280s loop, which in T state GP acts as a gate to control access to the catalytic site, is held in an open conformation in MalP. The open access to the conserved catalytic site provides an explanation for the activity without control in this basic archetype of a phosphorylase.