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Literature summary for 3.2.1.B40 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
Search for more literature for 3.2.1.B40

Activating Compound

Activating Compound Comment Organism Structure
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

Application Comment Organism
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)

Cloned (Comment) Organism
mutant enzyme E324G is expressed in Escherichia coli BL21(DE3) RIL Pyrococcus horikoshii
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

Protein Variants Comment Organism
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
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]

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

Localization

Localization Comment Organism GeneOntology No. Textmining
membrane bound to Pyrococcus horikoshii 16020
-
membrane membrane bound Pyrococcus horikoshii 16020
-

Organism

Organism UniProt Comment Textmining
Pyrococcus horikoshii O58104
-
-
Pyrococcus horikoshii OT-3 O58104
-
-

Purification (Commentary)

Purification (Comment) Organism
-
 Pyrococcus horikoshii
mutant enzyme E324G Pyrococcus horikoshii

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-nitrophenyl beta-D-glucopyranoside + H2O
-
 Pyrococcus horikoshii 2-nitrophenol + beta-D-glucopyranose
-
 ?
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
-
 ?
2-nitrophenyl beta-D-glucopyranoside + H2O
-
 Pyrococcus horikoshii OT-3 2-nitrophenol + beta-D-glucopyranose
-
 ?
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
-
 ?
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 ?
-
 ?
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

Subunits Comment Organism
monomer
-
 Pyrococcus horikoshii

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
75
-
 assay at Pyrococcus horikoshii

Turnover Number [1/s]

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

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
4
-
 assay at Pyrococcus horikoshii
4
-
 assay at, optimally active under acidic conditions Pyrococcus horikoshii

kcat/KM [mM/s]

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