Crystal structure and branching mechanism of GH57-type glycogen branching enzyme from Pyrococcus horikoshii

Abstract

Glycogen branching enzyme (GBE) is a branching enzyme that catalyzes the formation of α-1,6-branching points during glycogenesis by cleaving α-1,4 bond and making a new α-1,6 bond. Most GBEs belong to glycoside hydrolase 13 family, but new GBEs in the glycoside hydrolase 57 family (GH57) were isolated in archaebacteria. Here, I determine the crystal structure of a GH57 GBE from the hyperthermophilic archaebacteria, Pyrococcus horikoshii (PhGBE). The crystal of PhGBE belongs to P21 space group at a resolution of 2.3 Å. PhGBE has a central (β/α)7-barrel domain that contains an embedded helix domain, and an α-helix-rich C-terminal domain. The active-site cleft is located at the interface of the central domain and the C-terminal domain. Site-directed mutation was performed and the variant enzymes were checked. The iodine assay was applied to measure the enzymatic activities containing branching and hydrolase activities. The branching activity was measured by high-performance anion-exchange chromatography (HPAEC). This assay allows determination of the amount of branch points introduced by measuring the difference in the amount of reducing ends before and after debranching of the product by isoamylase. Mutation at Trp22, which is apart from the catalytic nucleophilic residue, abolished the both enzymatic activities, indicating that Trp22 might be responsible for the substrate recognition. I also observed that the deletion of a flexible loop near the catalytic residue changed the branching length of the product with an increased hydrolase activity. Taken together, these findings propose molecular mechanism for how the GH57 GBEs exhibit the two activities and where the substrates bind on the enzyme. These findings propose molecular mechanism for how the GH57-type GBEs exhibit the two activities and where the substrates bind on the enzyme. These researches may contribute to understanding how glycogen metabolism progresses in marine and extreme microorganisms and how glycogen metabolism plays a role in survival in the environments. In addition, this study will contribute to the use of GH57 GBE in the food industry.