title: Structural insight into the bifunctional mechanism of the glycogen-debranching enzyme TreX from the archaeon Sulfolobus solfataricus.
abstract: TreX is an archaeal glycogen-debranching enzyme that exists in two oligomeric states in solution, as a dimer and tetramer. Unlike its homologs, TreX from Sulfolobus solfataricus shows dual activities for alpha-1,4-transferase and alpha-1,6-glucosidase. To understand this bifunctional mechanism, we determined the crystal structure of TreX in complex with an acarbose ligand. The acarbose intermediate was covalently bound to Asp363, occupying subsites -1 to -3. Although generally similar to the monomeric structure of isoamylase, TreX exhibits two different active-site configurations depending on its oligomeric state. The N terminus of one subunit is located at the active site of the other molecule, resulting in a reshaping of the active site in the tetramer. This is accompanied by a large shift in the "flexible loop" (amino acids 399-416), creating connected holes inside the tetramer. Mutations in the N-terminal region result in a sharp increase in alpha-1,4-transferase activity and a reduced level of alpha-1,6-glucosidase activity. On the basis of geometrical analysis of the active site and mutational study, we suggest that the structural lid (acids 99-97) at the active site generated by the tetramerization is closely associated with the bifunctionality and in particular with the alpha-1,4-transferase activity. These results provide a structural basis for the modulation of activities upon TreX oligomerization that may represent a common mode of action for other glycogen-debranching enzymes in higher organisms.
authors.affiliation: Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, 111 Gwahangno, Yuseong-gu, Daejeon 305-806.
authors.forename: Eui-Jeon
authors.initials: EJ
authors.lastname: Woo
authors.forename: Seungjae
authors.initials: S
authors.lastname: Lee
authors.forename: Hyunju
authors.initials: H
authors.lastname: Cha
authors.forename: Jong-Tae
authors.initials: JT
authors.lastname: Park
authors.forename: Sei-Mee
authors.initials: SM
authors.lastname: Yoon
authors.forename: Hyung-Nam
authors.initials: HN
authors.lastname: Song
authors.forename: Kwan-Hwa
authors.initials: KH
authors.lastname: Park
mesh_terms: D020909:Acarbose / Q000737:chemistry; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D020134:Catalytic Domain; D004252:DNA Mutational Analysis; D005090:Exodeoxyribonucleases / Q000737:chemistry*; D006003:Glycogen / Q000737:chemistry*; D006004:Glycogen Debranching Enzyme System / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D048229:Sulfolobus solfataricus / Q000378:metabolism*
chemical_list: D006004:Glycogen Debranching Enzyme System; D010750:Phosphoproteins; D001224:Aspartic Acid; D006003:Glycogen; D005090:Exodeoxyribonucleases; C429526:three prime repair exonuclease 1; D020909:Acarbose