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Literature summary extracted from

  • Woo, E.J.; Lee, S.; Cha, H.; Park, J.T.; Yoon, S.M.; Song, H.N.; Park, K.H.
    Structural Insight into the Bifunctional Mechanism of the Glycogen-debranching Enzyme TreX from the Archaeon Sulfolobus solfataricus (2008), J. Biol. Chem., 283, 28641-28648.
    View publication on PubMedView publication on EuropePMC

Activating Compound

EC Number Activating Compound Comment Organism Structure
3.2.1.70 additional information 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 Saccharolobus solfataricus

Application

EC Number Application Comment Organism
3.2.1.70 additional information bifunctional mechanism of the archaeal glycogen-debranching enzyme TreX, showing alpha-1,4-transferase and alpha-1,6-glucosidase activities Saccharolobus solfataricus

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.4.1.25 expression of wild-type and mutant enzymes in Escherichia coli, sequence comparison Saccharolobus solfataricus
3.2.1.68 expression of wild-type and mutant enzymes in Escherichia coli, sequence comparison Saccharolobus solfataricus

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.4.1.25 TreX in complex with an acarbose ligand, microbatch method under oil at 18°C, dimeric crystal from 16% PEG 8000, 0.2 M NaCl, and 0.1 M CHES buffer, pH 9.5, tetrameric crystal form from 2.2 M ammonium phosphate and 0.1 M Tris-HCl buffer, pH 8.5. For the acarbose intermediate complex crystal, 0.1% acarbose is added to the protein, followed by incubation for 1 h prior to the setup of the crystal in 8% PEG 3000, 0.2M lithium sulfate, and 0.1 M imidazole buffer, pH 8.0, cyroprotection by 20% glycerol in mother liquor, in both crystal forms, the asymmetric unit consists of one dimer, X-ray diffraction structure determination and analysis at 2.8-3.0 A resolution Saccharolobus solfataricus
3.2.1.33 native dimer, native tetramer and the tetramer in complex with acarbose ligand covalently bound to residue D363, occupying subsites -1 to -3. Protein 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 of amino acids 399-416, creating connected holes inside the tetramer Saccharolobus solfataricus
3.2.1.68 TreX in complex with an acarbose ligand, microbatch method under oil at 18°C, dimeric crystal from 16% PEG 8000, 0.2 M NaCl, and 0.1 M CHES buffer, pH 9.5, tetrameric crystal form from 2.2 M ammonium phosphate and 0.1 M Tris-HCl buffer, pH 8.5. For the acarbose intermediate complex crystal, 0.1% acarbose is added to the protein, followed by incubation for 1 h prior to the setup of the crystal in 8% PEG 3000, 0.2M lithium sulfate, and 0.1 M imidazole buffer, pH 8.0, cyroprotection by 20% glycerol in mother liquor, in both crystal forms, the asymmetric unit consists of one dimer, X-ray diffraction structure determination and analysis at 2.8-3.0 A resolution Saccharolobus solfataricus
3.2.1.70 at a resolution of 2.8 A, TreX crystallized in the dimeric and tetrameric forms, in space groups P3121 and P321, respectively and in complex with an acarbose ligand. Acarbose intermediate is covalently bound to Asp363, occupying subsites -1 to -3 Saccharolobus solfataricus

Protein Variants

EC Number Protein Variants Comment Organism
2.4.1.25 additional information 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, overview Saccharolobus solfataricus
3.2.1.33 D318A sharp increasein alpha-1,4-transferase activity Saccharolobus solfataricus
3.2.1.33 E94A minor change in the amylo-1,6-glucosidase activity Saccharolobus solfataricus
3.2.1.68 additional information 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, overview Saccharolobus solfataricus
3.2.1.70 D318A shows an activity level identical to that of the wild-type Saccharolobus solfataricus
3.2.1.70 E94A shows a relatively minor change in the alpha-1,6-glucosidase activity, but a sharply increased alpha-1,4-transferase activity compared with the wild-type Saccharolobus solfataricus

Organism

EC Number Organism UniProt Comment Textmining
2.4.1.25 Saccharolobus solfataricus P95868
-
-
3.2.1.33 Saccharolobus solfataricus P95868 bifunctional amylo-1,6-glucosidase and 4-alpha-glucanotransferase
-
3.2.1.68 Saccharolobus solfataricus
-
-
-
3.2.1.70 Saccharolobus solfataricus
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.4.1.25 recombinant wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography Saccharolobus solfataricus
3.2.1.68 recombinant wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography Saccharolobus solfataricus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.4.1.25 additional information TreX from Sulfolobus solfataricus shows dual activities for alpha-1,4-transferase, EC 2.4.1.25 and alpha-1,6-glucosidase, EC 3.2.1.68, bifunctional mechanism, substrate maltotriose, overview. TreX exhibits two different active-site configurations depending on its oligomeric state Saccharolobus solfataricus ?
-
 ?
3.2.1.68 additional information TreX from Sulfolobus solfataricus shows dual activities for alpha-1,4-transferase, EC 2.4.1.25 and alpha-1,6-glucosidase, EC 3.2.1.68, bifunctional mechanism, substrate glycogen, overview. TreX exhibits two different active-site configurations depending on its oligomeric state Saccharolobus solfataricus ?
-
 ?
3.2.1.70 glycogen + H2O
-
 Saccharolobus solfataricus ?
-
 ?

Subunits

EC Number Subunits Comment Organism
2.4.1.25 dimer or tetramer the enzyme exists in two oligomeric states in solution, as a dimer and tetramer Saccharolobus solfataricus
2.4.1.25 More the structural lid, amino 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 Saccharolobus solfataricus
3.2.1.68 dimer or tetramer the enzyme exists in two oligomeric states in solution, as a dimer and tetramer Saccharolobus solfataricus
3.2.1.68 More the structural lid, amino 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 Saccharolobus solfataricus
3.2.1.70 dimer crystallography Saccharolobus solfataricus
3.2.1.70 tetramer crystallography Saccharolobus solfataricus

Synonyms

EC Number Synonyms Comment Organism
2.4.1.25 alpha-1,4-transferase
-
 Saccharolobus solfataricus
2.4.1.25 TreX
-
 Saccharolobus solfataricus
3.2.1.33 TreX
-
 Saccharolobus solfataricus
3.2.1.68 alpha-1,6-glucosidase
-
 Saccharolobus solfataricus
3.2.1.68 glycogen-debranching enzyme
-
 Saccharolobus solfataricus
3.2.1.68 TreX
-
 Saccharolobus solfataricus
3.2.1.70 alpha-1,6-glucosidase
-
 Saccharolobus solfataricus
3.2.1.70 TreX
-
 Saccharolobus solfataricus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.4.1.25 40
-
 assay at Saccharolobus solfataricus

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
3.2.1.68 40 70 assay at Saccharolobus solfataricus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.4.1.25 5.5
-
 assay at Saccharolobus solfataricus
3.2.1.68 5.5
-
 assay at Saccharolobus solfataricus