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Enzyme Nomenclature
Information on EC 3.2.1.B40 - Pyrococcus horikoshii beta-glycosidase
for references in articles please use BRENDA:EC3.2.1.B40preliminary BRENDA-supplied EC number
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3.2.1.B40 Pyrococcus horikoshii beta-glycosidaseSpecify your search results
The expected taxonomic range for this enzyme is: Pyrococcus horikoshii
Reaction Schemes
wide substrate specificity, high activity with beta-D-glucosides with long alkyl chains
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
4-nitrophenyl beta-D-galactopyranoside + H2O
4-nitrophenol + D-galactose
kcat/Km values of His-tagged enzyme decreasing in the order: beta-D-glucopyranoside, beta-D-galactopyranoside, beta-D-xylopyranoside, beta-D-mannopyranoside
-
-
?
4-nitrophenyl beta-D-glucopyranoside + H2O
4-nitrophenol + D-glucose
kcat/Km-values of His-tagged enzyme decreasing in the order: beta-D-glucopyranoside, beta-D-galactopyranoside, beta-D-xylopyranoside, beta-D-mannopyranoside
-
-
?
4-nitrophenyl beta-D-mannopyranoside + H2O
4-nitrophenol + D-mannose
kcat/Km values of His-tagged enzyme decreasing in the order: beta-D-glucopyranoside, beta-D-galactopyranoside, beta-D-xylopyranoside, beta-D-mannopyranoside
-
-
?
4-nitrophenyl beta-D-xylopyranoside + H2O
4-nitrophenol + D-xylose
kcat/Km values of His-tagged enzyme decreasing in the order: beta-D-glucopyranoside, beta-D-galactopyranoside, beta-D-xylopyranoside, beta-D-mannopyranoside
-
-
?
beta-D-Glc-(1->3)-beta-D-Glc + H2O
beta-D-glucose
i.e. laminaribiose, beta-linked glucose dimers are poorly hydrolyzed. The order of preference of His-tagged enzyme is beta-(1->3), beta-(1->4), beta-(1->6)
-
-
?
beta-D-Glc-(1->4)-beta-D-Glc + H2O
beta-D-glucose
i.e. cellobiose, beta-linked glucose dimers are poorly hydrolyzed. The order of preference of His-tagged enzyme is beta-(1->3), beta-(1->4), beta-(1->6)
-
-
?
beta-D-Glc-(1->6)-beta-D-Glc + H2O
beta-D-glucose
beta-linked glucose dimers are poorly hydrolyzed. The order of preference of His-tagged enzyme is beta-(1->3), beta-(1->4), beta-(1->6)
-
-
?
methyl beta-D-glucopyranoside + H2O
methanol + beta-D-glucose
-
-
-
?
n-amyl beta-D-glucopyranoside + H2O
1-pentanol + beta-D-glucose
-
-
-
?
n-decyl beta-D-glucopyranoside + H2O
1-decanol + beta-D-glucose
the best substrates for the His-tagged enzyme are beta-D-glucosides with long alkyl chains
-
-
?
n-dodecyl beta-D-glucopyranoside + H2O
1-dodecanol + beta-D-glucose
the best substrates for the His-tagged enzyme are beta-D-glucosides with long alkyl chains
-
-
?
n-hexyl beta-D-glucopyranoside + H2O
1-hexanol + beta-D-glucose
-
-
-
?
n-nonyl beta-D-glucopyranoside + H2O
1-nonanol + beta-D-glucose
-
-
-
?
n-octyl beta-D-glucopyranoside + H2O
1-octanol + beta-D-glucose
-
-
-
?
n-undecyl beta-D-glucopyranoside + H2O
1-undecanol + beta-D-glucose
the best substrates for the His-tagged enzyme are beta-D-glucosides with long alkyl chains
-
-
?
2-nitrophenyl beta-D-glucopyranoside + H2O
2-nitrophenol + beta-D-glucopyranose
-
-
-
?
2-nitrophenyl beta-D-glucopyranoside + H2O
2-nitrophenol + beta-D-glucopyranose
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
-
-
?
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
-
-
?
additional information
?
-
preferred substrate are alkyl beta-glycosides
-
-
?
additional information
?
-
the His-tagged enzyme has approximately 50% of the activity of the enzyme without a tag due to interference by the His-tag located at the N-terminus
-
-
?
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
15.11
2-nitrophenyl beta-D-glucopyranoside
pH 6.5, 50 mM sodium phosphate buffer + 3 M sodium formate, 75°C
30.55
2-nitrophenyl beta-D-glucopyranoside
pH 4.0, 50 mM sodium phosphate buffer, 75°C
39.47
2-nitrophenyl beta-D-glucopyranoside
pH 4.0, 200 mM sodium phosphate buffer, 75°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.092
2-nitrophenyl beta-D-glucopyranoside
pH 6.5, 50 mM sodium phosphate buffer + 3 M sodium formate, 75°C
0.592
2-nitrophenyl beta-D-glucopyranoside
pH 4.0, 50 mM sodium phosphate buffer, 75°C
1.302
2-nitrophenyl beta-D-glucopyranoside
pH 4.0, 200 mM sodium phosphate buffer, 75°C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.006
2-nitrophenyl beta-D-glucopyranoside
pH 6.5, 50 mM sodium phosphate buffer + 3 M sodium formate, 75°C
0.019
2-nitrophenyl beta-D-glucopyranoside
pH 4.0, 50 mM sodium phosphate buffer, 75°C
0.033
2-nitrophenyl beta-D-glucopyranoside
pH 4.0, 200 mM sodium phosphate buffer, 75°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). O58104_PYRHO
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
423
0
50330
TrEMBL
-
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
the enzyme is crystallized in the presence of a neutral surfactant, and the crystal structure is solved by the molecular replacement method and refined at 2.5 A. X-ray crystallography and docking simulation. The main-chain fold of the enzyme belongs to the (beta,alpha)8 barrel structure common to the family 1 glycosyl hydrolases. The active site is located at the center of the C-termini of the barrel beta-strands. The deep pocket of the active site accepts one sugar unit, and a hydrophobic channel extending radially from there binds the nonsugar moiety of the substrate. The docking simulation for oligosaccharides and alkylglucosides indicates that alkylglucosides with a long aliphatic chain are easily accommodated in the hydrophobic channel. This sparingly soluble enzyme has a cluster of hydrophobic residues on its surface, situated at the distal end of the active site channel and surrounded by a large patch of positively charged residues. It is proposed that this hydrophobic region can be inserted into the membrane while the surrounding positively charged residues make favorable contacts with phosphate groups on the inner surface of the membrane. The enzyme could thus adhere to the membrane in the proximity of its glycolipid substrate
the unusual structural features that confer the extreme thermostability and substrate preferences of this enzyme for long chain alkyl-beta-glycosides are investigated by X-ray crystallography and docking simulation. The enzyme is crystallized in the presence of a neutral surfactant, and the crystal structure is solved by the molecular replacement method and refined at 2.5 A. The main-chain fold of the enzyme belongs to the (betaalpha)8 barrel structure common to the Family 1 glycosyl hydrolases. The active site is located at the center of the C-termini of the barrel beta-strands. The deep pocket of the active site accepts one sugar unit, and a hydrophobic channel extending radially from there binds the nonsugar moiety of the substrate. The docking simulation for oligosaccharides and alkylglucosides indicated that alkylglucosides with a long aliphatic chain are easily accommodated in the hydrophobic channel
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
E324G
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
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
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
mutant enzyme E324G is expressed in Escherichia coli BL21(DE3) RIL
the mutant enzyme E324G is prepared as a fusion with an amino-terminal His-tag and expressed in Escherichia coli BL21(DE3) RIL strain
RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
denaturation with 8 M urea and the renaturation by direct dilution with buffer
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
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
Biocatal. Biotransform.
24
23-29
2006
Pyrococcus horikoshii (O58104), Pyrococcus horikoshii OT-3 (O58104)
-
Matsui, I.; Sakai, Y.; Matsui, E.; Kikuchi, H.; Kawarabayasi, Y.; Honda, K.
Novel substrate specificity of a membrane-bound beta-glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii
FEBS Lett.
467
195-200
2000
Pyrococcus horikoshii (O58104), Pyrococcus horikoshii OT-3 (O58104)
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Release 2023.1 (January 2023)
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