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699 a.a.
Key reference
DOI no: 10.1074/jbc.M802560200 J Biol Chem 283:28641-28648 (2008) PubMed id: 18703518
Structural insight into the bifunctional mechanism of the glycogen-debranching enzyme TreX from the archaeon Sulfolobus solfataricus. E.J.Woo, S.Lee, H.Cha, J.T.Park, S.M.Yoon, H.N.Song, K.H.Park. 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.
Selected figure(s)
Figure 3.
Active site with the acarbose covalent intermediate. A, the acarbose intermediate covalently bound to Asp^363 shows tight interactions with the residues at subsite -1 in the dimer. The inhibitory acarviosine moiety is bound to subsites -2 and -3. The catalytic residues (Asp^363, Glu^399, and Asp^471) and other conserved residues in the active site are indicated by sticks. The ligand molecules are shown in yellow. Helix α4 unique to TreX is in pink. B, the electron density map of 2F[o] - F[c] (1.0 σ) shows the acarbose intermediate molecule at subsites -1, -2, and -3 (left) and the glucose molecules at subsites +2 and +3 (right). C, the omit map of F[o] - F[c] drawn without the ligand shows the covalent binding of the acarbose intermediate to Asp^326, contoured at 3.0 σ (left) and 5.0 σ (right).Figure 4.
Reshaped active site in the tetrameric structure. A, the substrate cleft is covered with lid 1 (aa 92-97; purple) and lid 2 (aa 315-322; cyan) generated by the tetrameric arrangement. The N-terminal domain is shown in blue. The glucose and acarbose intermediate ligands in the complex structure are superposed and indicated by sticks. The flexible loop (aa 399-416) is orange. B, the conformational change in the flexible loop results in a shift of Glu^399 and Tyr^408 away from the other catalytic residues Asp^363 and Asp^471. The region in the comparison is blue for the dimeric structure and orange for the tetramer. C, the active sites (black circles) highlighted by the superposed ligands are seen along the connected substrate-binding grooves inside the tetramer. mole, molecule.The above figures are reprinted from an Open Access publication published by the ASBMB: J Biol Chem (2008, 283, 28641-28648) copyright 2008. Figures were selected by an automated process.
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