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

학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부, 2017. 2. 하남출.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.I. INTRODUCTION 1 II. MATERIALS AND METHODS 5 2.1. Plasmid construction 5 2.2. Site direct mutagenesis 5 2.3. Purification of GBE and variant GBE proteins 6 2.3.1. Overexpression 6 2.3.2. Affinity chromatography 7 2.3.3. Anion-exchange chromatography 7 2.3.4. Size exclusion chromatography 7 2.4. Crystallization 8 2.5. Structure determination and refinement 8 2.6. Iodine assay 9 2.7. Branching assay 10 III. RESULTS 13 3.1. Overexpression and purification of PhGBE and variant PhGBE proteins 13 3.2. Crystal of PhGBE 18 3.3. Structural determination of PhGBE 19 3.4. Overall structure of PhGBE 21 3.5. Structural comparison with other GH57 proteins 24 3.6. The active site of PhGBE 29 3.7. The role of the flexible loop in the catalysis 35 IV. DISCUSSION 39 V. REFERENCES 45 VI. 국문초록 52Maste