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Literature summary extracted from

  • Perugino, G.; Falcicchio, P.; Michela Corsaro, M.; Matsui, I.; Parrilli, M.; Rossi, M.; Moracci, M.
    Preparation of a glycosynthase from the beta-glycosidase of the Archaeon Pyrococcus horikoshii (2006), Biocatal. Biotransform., 24, 23-29 .
No PubMed abstract available

Activating Compound

EC Number Activating Compound Comment Organism Structure
3.2.1.B40 formate the mutant enzyme does not have any activity with the substrate 2-nitrophenyl-beta-D-glucopyranose. However, in the presence of 3 M sodium formate the mutant is reactivated, 10fold increment between 0.1 M and 3 M sodium formate Pyrococcus horikoshii

Application

EC Number Application Comment Organism
3.2.1.B40 synthesis in the presence of sodium formate buffer pH 4.0 at 75°C the E324G mutant acts as a hyperthermophilic glycosynthase. Though the yield of the reaction does not exceed 10%, it is demonstrated that this could be a general strategy for the preparation of hyperthermophilic glycosynthase. The peculiar specificity of the enzyme for alkyl-glycosides makes the resulting glycosynthase a promising tool for biocatalysis Pyrococcus horikoshii

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.2.1.B40 mutant enzyme E324G is expressed in Escherichia coli BL21(DE3) RIL Pyrococcus horikoshii
3.2.1.B40 the mutant enzyme E324G is prepared as a fusion with an amino-terminal His-tag and expressed in Escherichia coli BL21(DE3) RIL strain Pyrococcus horikoshii

Protein Variants

EC Number Protein Variants Comment Organism
3.2.1.B40 E324G mutant enzyme does not have any activity with the substrate 2-nitrophenyl-beta-D-glucopyranose. However, in the presence of 3 M sodium formate the mutant is reactivated. acts as a hyperthermophilic glycosynthase. kcat/KM of the mutant enzyme is more than 5-fold higher at pH 4.0 than that at pH 6.5. The hydrolytic activity of the mutant at 90°C at pH 6.5 in 3 M sodium formate is about 600fold lower than that of the wild type assayed on 4-nitrophenyl-glucose at 90°C in 50 mM sodium phosphate buffer pH 6.0, 0.1% Triton X-100, and 0.3 M NaCl Pyrococcus horikoshii
3.2.1.B40 E324G mutation completely eliminates the activity of the enzyme. Activity can be reactivated in sodium formate at pH 4.0. The enzyme acts as a glycosynthase. The peculiar specificity of the mutant enzyme E324G for alkyl-glycosides makes the glycosynthase a promising tool for biocatalysis Pyrococcus horikoshii

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.2.1.B40 15.11
-
 2-nitrophenyl beta-D-glucopyranoside pH 6.5, 50 mM sodium phosphate buffer + 3 M sodium formate, 75°C Pyrococcus horikoshii
3.2.1.B40 30.55
-
 2-nitrophenyl beta-D-glucopyranoside pH 4.0, 50 mM sodium phosphate buffer, 75°C Pyrococcus horikoshii
3.2.1.B40 39.47
-
 2-nitrophenyl beta-D-glucopyranoside pH 4.0, 200 mM sodium phosphate buffer, 75°C Pyrococcus horikoshii

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
3.2.1.B40 membrane bound to Pyrococcus horikoshii 16020
-
3.2.1.B40 membrane membrane bound Pyrococcus horikoshii 16020
-

Organism

EC Number Organism UniProt Comment Textmining
3.2.1.B40 Pyrococcus horikoshii O58104
-
-
3.2.1.B40 Pyrococcus horikoshii OT-3 O58104
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.2.1.B40
-
 Pyrococcus horikoshii
3.2.1.B40 mutant enzyme E324G Pyrococcus horikoshii

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.2.1.B40 2-nitrophenyl beta-D-glucopyranoside + H2O
-
 Pyrococcus horikoshii 2-nitrophenol + beta-D-glucopyranose
-
 ?
3.2.1.B40 2-nitrophenyl beta-D-glucopyranoside + H2O wide substrate specificity belonging to family 1 of glycoside hydrolases classification. The mutant enzyme E324G does not have any activity with the substrate 2-nitrophenyl-beta-D-glucopyranose. However, in the presence of 3 M sodium formate the mutant is reactivated Pyrococcus horikoshii 2-nitrophenol + beta-D-glucopyranose
-
 ?
3.2.1.B40 2-nitrophenyl beta-D-glucopyranoside + H2O
-
 Pyrococcus horikoshii OT-3 2-nitrophenol + beta-D-glucopyranose
-
 ?
3.2.1.B40 2-nitrophenyl beta-D-glucopyranoside + H2O wide substrate specificity belonging to family 1 of glycoside hydrolases classification. The mutant enzyme E324G does not have any activity with the substrate 2-nitrophenyl-beta-D-glucopyranose. However, in the presence of 3 M sodium formate the mutant is reactivated Pyrococcus horikoshii OT-3 2-nitrophenol + beta-D-glucopyranose
-
 ?
3.2.1.B40 additional information in the presence of sodium formate buffer pH 4.0 at 75°C the E324G mutant acts as a hyperthermophilic glycosynthase. Though the yield of the reaction does not exceed 10%, it is demonstrated that this could be a general strategy for the preparation of hyperthermophilic glycosynthase Pyrococcus horikoshii ?
-
 ?
3.2.1.B40 additional information in the presence of sodium formate buffer pH 4.0 at 75°C the E324G mutant acts as a hyperthermophilic glycosynthase. Though the yield of the reaction does not exceed 10%, it is demonstrated that this could be a general strategy for the preparation of hyperthermophilic glycosynthase Pyrococcus horikoshii OT-3 ?
-
 ?

Subunits

EC Number Subunits Comment Organism
3.2.1.B40 monomer
-
 Pyrococcus horikoshii

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.2.1.B40 75
-
 assay at Pyrococcus horikoshii

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.2.1.B40 0.092
-
 2-nitrophenyl beta-D-glucopyranoside pH 6.5, 50 mM sodium phosphate buffer + 3 M sodium formate, 75°C Pyrococcus horikoshii
3.2.1.B40 0.592
-
 2-nitrophenyl beta-D-glucopyranoside pH 4.0, 50 mM sodium phosphate buffer, 75°C Pyrococcus horikoshii
3.2.1.B40 1.302
-
 2-nitrophenyl beta-D-glucopyranoside pH 4.0, 200 mM sodium phosphate buffer, 75°C Pyrococcus horikoshii

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.2.1.B40 4
-
 assay at Pyrococcus horikoshii
3.2.1.B40 4
-
 assay at, optimally active under acidic conditions Pyrococcus horikoshii

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.2.1.B40 0.006
-
 2-nitrophenyl beta-D-glucopyranoside pH 6.5, 50 mM sodium phosphate buffer + 3 M sodium formate, 75°C Pyrococcus horikoshii
3.2.1.B40 0.019
-
 2-nitrophenyl beta-D-glucopyranoside pH 4.0, 50 mM sodium phosphate buffer, 75°C Pyrococcus horikoshii
3.2.1.B40 0.033
-
 2-nitrophenyl beta-D-glucopyranoside pH 4.0, 200 mM sodium phosphate buffer, 75°C Pyrococcus horikoshii