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Literature summary extracted from

  • Park, K.H.; Jung, J.H.; Park, S.G.; Lee, M.E.; Holden, J.F.; Park, C.S.; Woo, E.J.
    Structural features underlying the selective cleavage of a novel exo-type maltose-forming amylase from Pyrococcus sp. ST04 (2014), Acta Crystallogr. Sect. D, 70, 1659-1668.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.2.1.B36 recombinant expression of selenomethionine-labeled wild-type and mutant enzymes in Escherichia coli strain BL21-CodonPlus(DE3)-RP Pyrococcus sp.
3.2.1.133 expressed in Escherichia coli BL21-CodonPlus(DE3)-RP cells Pyrococcus sp.

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.2.1.B36 purified recombinant selenomethionine-labeled wild-type enzyme, sitting drop vapour diffusion method, mixing of 0.001 ml of 15 mg/ml protein solution with 0.001 ml of reservoir solution consisting of 1.0 M ammonium citrate dibasic, 0.8 M sodium acetate trihydrate pH 4.6, 18°C, X-ray diffraction structure determination and analysis at 1..8 A resolution Pyrococcus sp.
3.2.1.133 sitting drop vapor diffusion method, using 1.0 M ammonium citrate dibasic, 0.8 M sodium acetate trihydrate pH 4.6 Pyrococcus sp.

Protein Variants

EC Number Protein Variants Comment Organism
3.2.1.B36 D253N site-directed mutagenesis, the mutant shows reduced alpha-1,4- and alpha1,6-hydrolytic activity compared to the wild-type enzyme Pyrococcus sp.
3.2.1.B36 E153Q site-directed mutagenesis, the mutant shows reduced alpha-1,4- and alpha1,6-hydrolytic activity compared to the wild-type enzyme Pyrococcus sp.
3.2.1.B36 E580Q site-directed mutagenesis, the mutant shows reduced alpha-1,4- and alpha1,6-hydrolytic activity compared to the wild-type enzyme Pyrococcus sp.
3.2.1.B36 F218A site-directed mutagenesis, the mutant exhibits 3.5fold icreased alpha-1,4-glucosidic bond hydrolysis compared to the wild-type enzyme Pyrococcus sp.
3.2.1.B36 F452A site-directed mutagenesis, the mutant shows reduced alpha-1,4- and alpha1,6-hydrolytic activity compared to the wild-type enzyme Pyrococcus sp.
3.2.1.B36 W453A site-directed mutagenesis, the mutant shows reduced alpha-1,4- and alpha1,6-hydrolytic activity compared to the wild-type enzyme Pyrococcus sp.
3.2.1.133 E580Q strongly reduced activity compared to the wild type enzyme Pyrococcus sp.
3.2.1.133 F218A the mutant shows wild type activity with alpha-1,6-glycosidic bond hydrolysis and about 4fold increased activity with alpha-1,4-glycosidic bond hydrolysis compared to the wild type enzyme Pyrococcus sp.
3.2.1.133 F452A inactive Pyrococcus sp.
3.2.1.133 W453A strongly reduced activity compared to the wild type enzyme Pyrococcus sp.

Inhibitors

EC Number Inhibitors Comment Organism Structure
3.2.1.133 acarbose about 80% residual activity at 1 mM Pyrococcus sp.
3.2.1.133 maltose about 75% residual activity at 1 mM Pyrococcus sp.
3.2.1.133 additional information not inhibited by panose and isomaltose Pyrococcus sp.

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.2.1.B36 additional information no metal ions Pyrococcus sp.

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
3.2.1.B36 70191
-
 2 * 70191, recombinant enzyme, SDS-PAGE, in the dimeric structure, extensive interactions including Asn185-Gly327, Glu224-Lys241 and Gly327-Asn185 hydrogen bonds and a Glu224-Lys241 salt bridge between two (beta/alpha)7-barrels of the N-domains comprise the dimer associations Pyrococcus sp.
3.2.1.B36 260000
-
 recombinant enzyme, gel filtration Pyrococcus sp.

Organism

EC Number Organism UniProt Comment Textmining
3.2.1.B36 Pyrococcus sp. I3RE04
-
-
3.2.1.B36 Pyrococcus sp. ST04 I3RE04
-
-
3.2.1.133 Pyrococcus sp. I3RE04
-
-
3.2.1.133 Pyrococcus sp. ST04 I3RE04
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.2.1.B36 recombinant wild-type and mutant enzymes from Escherichia coli strain BL21-CodonPlus(DE3)-RP Pyrococcus sp.
3.2.1.133
-
 Pyrococcus sp.

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.2.1.B36 additional information the enzyme hydrolyzes both alpha-1,4-glucosidic and alpha-1,6-glucosidic linkages of substrates, recognizing only maltose units, in an exo-type manner. Substrate specificity and binding, docking modelling, overview Pyrococcus sp. ?
-
 ?
3.2.1.B36 additional information the enzyme hydrolyzes both alpha-1,4-glucosidic and alpha-1,6-glucosidic linkages of substrates, recognizing only maltose units, in an exo-type manner. Substrate specificity and binding, docking modelling, overview Pyrococcus sp. ST04 ?
-
 ?
3.2.1.133 maltotriose + H2O
-
 Pyrococcus sp. maltose + D-glucose
-
 ?
3.2.1.133 maltotriose + H2O
-
 Pyrococcus sp. ST04 maltose + D-glucose
-
 ?
3.2.1.133 additional information acarbose is not cleaved by the enzyme Pyrococcus sp. ?
-
 ?
3.2.1.133 additional information the enzyme hydrolyzes both alpha-1,4-glucosidic and alpha-1,6-glucosidic linkages of substrates, recognizing only maltose units, in an exo-type manner Pyrococcus sp. ?
-
 ?
3.2.1.133 additional information acarbose is not cleaved by the enzyme Pyrococcus sp. ST04 ?
-
 ?
3.2.1.133 additional information the enzyme hydrolyzes both alpha-1,4-glucosidic and alpha-1,6-glucosidic linkages of substrates, recognizing only maltose units, in an exo-type manner Pyrococcus sp. ST04 ?
-
 ?

Subunits

EC Number Subunits Comment Organism
3.2.1.B36 dimer 2 * 70191, recombinant enzyme, SDS-PAGE, in the dimeric structure, extensive interactions including Asn185-Gly327, Glu224-Lys241 and Gly327-Asn185 hydrogen bonds and a Glu224-Lys241 salt bridge between two (beta/alpha)7-barrels of the N-domains comprise the dimer associations Pyrococcus sp.
3.2.1.B36 More the enzyme structure shows a tight ring-shaped tetramer with monomers composed of two domains: an N-domain (amino acids 1-341) with a typical GH57 family (beta/alpha)7-barrel fold and a C-domain (amino acids 342-597) composed of alpha-helical bundles, two domains assemble to form a groove in the active site Pyrococcus sp.
3.2.1.133 monomer
-
 Pyrococcus sp.

Synonyms

EC Number Synonyms Comment Organism
3.2.1.B36 maltose-forming alpha-amylase
-
 Pyrococcus sp.
3.2.1.B36 PSMA
-
 Pyrococcus sp.
3.2.1.133 maltose-forming alpha-amylase
-
 Pyrococcus sp.

General Information

EC Number General Information Comment Organism
3.2.1.B36 evolution the enzyme belongs to the glycoside hydrolase family 57, GH57 Pyrococcus sp.
3.2.1.B36 additional information the enzyme structure shows a tight ring-shaped tetramer with monomers composed of two domains: an N-domain (amino acids 1-341) with a typical GH57 family (beta/alpha)7-barrel fold and a C-domain (amino acids 342-597) composed of alpha-helical bundles. The catalytic residues are Glu153 and Asp253 at the domain interface Pyrococcus sp.