Information on EC 3.2.1.140 - lacto-N-biosidase

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The expected taxonomic range for this enzyme is: Actinobacteridae

EC NUMBER
COMMENTARY
3.2.1.140
-
RECOMMENDED NAME
GeneOntology No.
lacto-N-biosidase
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
beta-D-Gal-(1->3)-beta-D-GlcNAc-(1->3)-beta-D-Gal-(1->4)-D-Glc + H2O = beta-D-Gal-(1->3)-D-GlcNAc + beta-D-Gal-(1->4)-D-Glc
show the reaction diagram
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-
-
-
beta-D-Gal-(1->3)-beta-D-GlcNAc-(1->3)-beta-D-Gal-(1->4)-D-Glc + H2O = beta-D-Gal-(1->3)-D-GlcNAc + beta-D-Gal-(1->4)-D-Glc
show the reaction diagram
catalytic reaction via Michaelis complex, transition state 1, oxazolinium ion intermediate, and transition state 2, overview
beta-D-Gal-(1->3)-beta-D-GlcNAc-(1->3)-beta-D-Gal-(1->4)-D-Glc + H2O = beta-D-Gal-(1->3)-D-GlcNAc + beta-D-Gal-(1->4)-D-Glc
show the reaction diagram
catalytic reaction via Michaelis complex, transition state 1, oxazolinium ion intermediate, and transition state 2, overview
Bifidobacterium bifidum JCM1254
-
-
lacto-N-tetraose + H2O = lacto-N-biose + lactose
show the reaction diagram
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-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
condensation
lacto-N-biose + lactose + enzyme
condensation
Bifidobacterium bifidum JCM1254
-
lacto-N-biose + lactose + enzyme
-
hydrolysis
Bifidobacterium bifidum JCM1254
-
-
-
hydrolysis of O-glycosyl bond
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-
-
-
transglycosylation
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transglycosylation
Bifidobacterium bifidum JCM1254
-
-
-
SYSTEMATIC NAME
IUBMB Comments
oligosaccharide lacto-N-biosylhydrolase
The enzyme from Streptomyces specifically hydrolyses the terminal lacto-N-biosyl residue (beta-D-Gal-(1->3)-D-GlcNAc) from the non-reducing end of oligosaccharides with the structure beta-D-Gal-(1->3)-beta-D-GlcNAc-(1->3)-beta-D-Gal-(1->R). Lacto-N-hexaose (beta-D-Gal-(1->3)-beta-D-GlcNAc-(1->3)-beta-D-Gal-(1->3)-beta-D-GlcNAc-(1->3)-beta-D-Gal-(1->4)-D-Glc) is hydrolysed to form first lacto-N-tetraose plus lacto-N-biose, with the subsequent formation of lactose. Oligosaccharides in which the non-reducing terminal Gal or the penultimate GlcNAc are replaced by fucose or sialic acid are not substrates. Asialo GM1 tetraose (beta-D-Gal-(1->3)-beta-D-GalNAc-(1->3)-beta-D-Gal-(1->4)-D-Glc) is hydrolysed very slowly, but lacto-N-neotetraose (beta-D-Gal-(1->4)-beta-D-GalNAc-(1->3)-beta-D-Gal-(1->4)-D-Glc) is not a substrate
SYNONYMS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
BLLJ_1505
Bifidobacterium longum JCM1217
-
-
BLLJ_1506
Bifidobacterium longum JCM1217
-
-
lacto-N-biosidase
-
-
-
-
lacto-N-biosidase
-
-
lacto-N-biosidase
-
lacto-N-biosidase
Bifidobacterium bifidum JCM1254
-
-
lacto-N-biosidase
-
-
lacto-N-biosidase (Streptomyces strain 142)
-
-
-
-
LNBase
Bifidobacterium bifidum JCM1254
-
-
LnbB
Bifidobacterium bifidum JCM1254
-
-
CAS REGISTRY NUMBER
COMMENTARY
146359-52-6
-
183511-11-7
lacto-N-biosidase (Streptomyces strain 142)
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain JCM1254 with highest activity, taken for gene sequencing and further characterization, also activity in strains JCM1255, and JCM7004
UniProt
Manually annotated by BRENDA team
lacto-N-biosidase activity is found in several bifidobacterial strains: strains Bifidobacterium bifidum JCM 1255 and JCM 7004, Bifidobacterium longum JCM 1217, JCM 1222, JCM 7045, but not in the other enteric bacteria, such as clostridia, bacteroides, and lactobacilli
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-
Manually annotated by BRENDA team
Bifidobacterium bifidum JCM1254
gene lnbB
UniProt
Manually annotated by BRENDA team
Bifidobacterium bifidum JCM1254
strain JCM1254 with highest activity, taken for gene sequencing and further characterization, also activity in strains JCM1255, and JCM7004
UniProt
Manually annotated by BRENDA team
enzyme activity in strains JCM1217, JCM1222, JCM7054, found in infant intestines; lacto-N-biosidase activity is found in several bifidobacterial strains: strains Bifidobacterium bifidum JCM 1254, JCM 1255, and JCM 7004, Bifidobacterium longum JCM 1217, JCM 1222, and JCM 7045, but not in the other enteric bacteria, such as clostridia, bacteroides, and lactobacilli
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-
Manually annotated by BRENDA team
subsp. longum, gene LnbX and LnbY
UniProt
Manually annotated by BRENDA team
Bifidobacterium longum JCM1217
subsp. longum, gene LnbX and LnbY
UniProt
Manually annotated by BRENDA team
strain L-101
-
-
Manually annotated by BRENDA team
Microbacterium sp. L-101
strain L-101
-
-
Manually annotated by BRENDA team
no activity in Bifidobacterium breve
strains found in infant intestines, bacteria grown anaerobically in basal lactose medium, 37 degrees Celsius, enzyme assay 3 h - overnight, in 100 mM potassium phosphate buffer pH 7.0 and 4 mM substrate lacto-N-tetraose, HPLC with absorbance measurement of N-acetyl group at 214 nm
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-
Manually annotated by BRENDA team
no activity in Bifidobacterium catenulatum
strains found in infant intestines, bacteria grown anaerobically in basal lactose medium, 37 degrees Celsius, enzyme assay 3 h - overnight, in 100 mM potassium phosphate buffer pH 7.0 and 4 mM substrate lacto-N-tetraose, HPLC with absorbance measurement of N-acetyl group at 214 nm
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-
Manually annotated by BRENDA team
strain 142
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-
Manually annotated by BRENDA team
Streptomyces sp. 142
strain 142
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
the enzyme belongs to the glycoside hydrolase family 20, GH20
evolution
the LnbX protein is found only in Bifidobacterium bifidum, Bifidobacterium longum, and a few gut microbes, suggesting that the protein has evolved in specialized niches
evolution
Bifidobacterium bifidum JCM1254
-
the enzyme belongs to the glycoside hydrolase family 20, GH20
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evolution
Bifidobacterium longum JCM1217
-
the LnbX protein is found only in Bifidobacterium bifidum, Bifidobacterium longum, and a few gut microbes, suggesting that the protein has evolved in specialized niches
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metabolism
-
the enzyme is critical in the lacto-N-biose pathway
metabolism
the enzyme liberates lacto-N-biose I, i.e. Gal-beta-(1->3)-GlcNAc, the major core structure, from the nonreducing end of human milk oligosaccharides and plays a key role in the metabolic pathway of these compounds
physiological function
the enzyme liberates lacto-N-biose I, the major core structure, from the nonreducing end of human milk oligosaccharides and plays a key role in the metabolic pathway of these compounds
physiological function
Bifidobacterium bifidum JCM1254
-
the enzyme liberates lacto-N-biose I, the major core structure, from the nonreducing end of human milk oligosaccharides and plays a key role in the metabolic pathway of these compounds
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metabolism
Bifidobacterium bifidum JCM1254
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the enzyme liberates lacto-N-biose I, i.e. Gal-beta-(1->3)-GlcNAc, the major core structure, from the nonreducing end of human milk oligosaccharides and plays a key role in the metabolic pathway of these compounds
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additional information
-
three realistic mechanistic alternatives exist for lacto-N-biosidases: substrate-assisted catalytic mechanism, or a mechanism involving the formation and breakdown of a covalent aglycosyl enzyme intermediate, or an inverting mechanism, overview. A key difference between these mechanistic alternatives is the involvement of the 2-acetamido group of the substrate. The lacto-N-biosidase-type enzyme uses a mechanism involving substrate-assisted catalysis from the 2-acetamido group of the substrate. Decrease in second-order rate constant, the carbonyl oxygen acts as a nucleophile, attacking the anomeric centre
additional information
the enzyme reaction proceeds via a substrate-assisted catalytic mechanism. The enzyme consists of three domains, and the C-terminal domain has a unique beta-trefoil-like fold. Compared with other beta-N-acetylhexosaminidases, lacto-N-biosidase has a wide substrate-binding pocket with a -2 subsite specific for beta-1,3-linked Gal, three-dimensional structure and possible conformational pathway for the lacto-N-biosidase reaction, overview. Enzyme structure and active site structure comparisons. The two catalytic residues of GH20, Asp320 (polarizing residue) and Glu321 (acid/base catalytic residue), form hydrogen bonds with the amide nitrogen of the 2-acetamido group and the O1-hydroxyl,respectively. Tyr-419 is a highly conserved residue in GH20 enzymes, and its side-chain hydroxyl group forms a hydrogen bond with the carbonyl oxygen atom of the 2-acetamido group. Asp467 forms bifurcated hydrogen bonds with the O4- and O6-hydroxyl groups of the GlcNAc residue. Catalytic reaction mechanism and conformational changes analysis
additional information
Bifidobacterium bifidum JCM1254
-
the enzyme reaction proceeds via a substrate-assisted catalytic mechanism. The enzyme consists of three domains, and the C-terminal domain has a unique beta-trefoil-like fold. Compared with other beta-N-acetylhexosaminidases, lacto-N-biosidase has a wide substrate-binding pocket with a -2 subsite specific for beta-1,3-linked Gal, three-dimensional structure and possible conformational pathway for the lacto-N-biosidase reaction, overview. Enzyme structure and active site structure comparisons. The two catalytic residues of GH20, Asp320 (polarizing residue) and Glu321 (acid/base catalytic residue), form hydrogen bonds with the amide nitrogen of the 2-acetamido group and the O1-hydroxyl,respectively. Tyr-419 is a highly conserved residue in GH20 enzymes, and its side-chain hydroxyl group forms a hydrogen bond with the carbonyl oxygen atom of the 2-acetamido group. Asp467 forms bifurcated hydrogen bonds with the O4- and O6-hydroxyl groups of the GlcNAc residue. Catalytic reaction mechanism and conformational changes analysis
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SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-fucosyllactose + H2O
lactose + fucose
show the reaction diagram
-
Fuc-alpha-(1->2)-Gal-beta-(1->4)-Glc
-
?
4-nitrophenyl-GalNAc-beta-(1->3)-GlcNAc + H2O
4-nitrophenol + GalNAc-beta-(1->3)-GlcNAc
show the reaction diagram
-
-
?
4-nitrophenyl-lacto-N-bioside + H2O
4-nitrophenol + lacto-N-biose
show the reaction diagram
-
-
?
D-Gal-(1,3)-beta-GlcNAc-(1,3)-beta-D-Gal-(1,4)-beta-D-Glc-pyridylamine + H2O
?
show the reaction diagram
Bifidobacterium bifidum, Bifidobacterium bifidum JCM1254
lacto-N-tetraose-pyridylamine, no activity on alpha-linked disaccharides, beta-linked p-nitrophenyl monosaccarides such as p-nitrophenyl-beta-BlcNAc and p-nitrophenyl-beta-GalNAc, no activity on ganglioside GA1 structure with beta-linked galacto-N-biose, no activity on fucosylated forms of lacto-N-tetraose or lacto-N-neotetraose
-
-
?
D-Gal-(1,3)-beta-GlcNAc-(1,3)-beta-D-Gal-(1,4)-D-Glc + H2O
lactose + D-galactosyl-beta-1,3-N-acetyl-D-glucosamine
show the reaction diagram
-
lacto-N-tetraose, major component of human milk oligosaccharides
lacto-N-biose I
?
D-Gal-(1,3)-beta-GlcNAc-(1,3)-beta-D-Gal-(1,4)-D-Glc + H2O
lactose + D-galactosyl-beta-1,3-N-acetyl-D-glucosamine
show the reaction diagram
Bifidobacterium bifidum, Bifidobacterium bifidum JCM1254
lacto-N-tetraose, major component of human milk oligosaccharides
lacto-N-biose I
?
Galbeta1-3GlcNAcbeta-p-nitrophenol + H2O
Galbeta1-3GlcNAc + p-nitrophenol
show the reaction diagram
Microbacterium sp., Microbacterium sp. L-101
-
-
-
?
Galbeta1-3GlcNAcbeta-p-nitrophenol + lactose
Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc + ?
show the reaction diagram
Microbacterium sp., Microbacterium sp. L-101
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-
Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc, i.e. lacto-neotetraose
?
lacto-N-difucohexaose + H2O
lactose + Fuc-alpha-(1->2)-Gal-beta-(1->3)[Fuc-alpha-(1->4)]GlcNAc
show the reaction diagram
-
Fuc-alpha-(1->2)Gal-beta-(1->3)[Fuc-alpha-(1->4)]GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc
-
?
lacto-N-fucopentaose I + H2O
lactose + Fuc-alpha-(1->2)-Gal-beta-(1->3)-GlcNAc
show the reaction diagram
-
Fuc-alpha-(1->2)-Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc
-
?
lacto-N-fucopentaose I + H2O
lactose + Fuc-alpha-(1->2)-Gal-beta-(1->3)-GlcNAc
show the reaction diagram
Bifidobacterium longum, Bifidobacterium longum JCM1217
Fuc-alpha-(1->2)-Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc
-
?
lacto-N-hexaose + H2O
lactose + lacto-N-tetraose
show the reaction diagram
Gal-beta-(1->3)-GlcNAc-beta-(1->3)-[Gal-beta-(1->4)-GlcNAc-beta-(1->6)]-beta-(1->4)-Glc
-
?
lacto-N-tetraose + H2O
lacto-N-biose
show the reaction diagram
-
-
-
-
lacto-N-tetraose + H2O
lacto-N-biose
show the reaction diagram
-
lacto-N-tetraose is a major component of human milk oligosaccharides, substrate used to examine the LnbB activities in various bacterial strains
-
?
lacto-N-tetraose + H2O
lacto-N-biose
show the reaction diagram
Streptomyces sp. 142
-
-
-
-
lacto-N-tetraose + H2O
lactose + lacto-N-biose
show the reaction diagram
-
Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc
-
?
p-nitrophenyl-Gal-(1,3)-beta-GalNAc + H2O
?
show the reaction diagram
Bifidobacterium bifidum, Bifidobacterium bifidum JCM1254
30% enzyme activity compared to p-nitrophenyl-Gal-(1,3)-beta-GlcNAc
-
-
?
p-nitrophenyl-Gal-(1,3)-beta-GlcNAc + H2O
?
show the reaction diagram
lacto-N-biosyl-beta-p-nitrophenyl, more genereal lacto-N-tetraose with 1-alcanols (methanol, ethanol, 1-propanol, 1-butanol) or p-nitrophenyl-beta-lacto-N-biose + lactose with enzyme
-
-
?
p-nitrophenyl-Gal-(1,3)-beta-GlcNAc + H2O
?
show the reaction diagram
-
30% relative activity compared to p-nitrophenyl-lacto-N-bioside
-
-
?
p-nitrophenyl-Gal-(1,3)-beta-GlcNAc + H2O
?
show the reaction diagram
Bifidobacterium bifidum JCM1254
lacto-N-biosyl-beta-p-nitrophenyl, more genereal lacto-N-tetraose with 1-alcanols (methanol, ethanol, 1-propanol, 1-butanol) or p-nitrophenyl-beta-lacto-N-biose + lactose with enzyme
-
-
?
p-nitrophenyl-lacto-N-bioside + H2O
p-nitrophenol + lacto-N-biose
show the reaction diagram
-
-
-
-
p-nitrophenyl-lacto-N-bioside + H2O
p-nitrophenol + lacto-N-biose
show the reaction diagram
-
-
-
-
p-nitrophenyl-lacto-N-bioside + H2O
p-nitrophenol + lacto-N-biose
show the reaction diagram
-
most active substrate
-
?
p-nitrophenyl-lacto-N-bioside + H2O
p-nitrophenol + lacto-N-biose
show the reaction diagram
Streptomyces sp. 142
-
-
-
-
p-nitrophenyl-lacto-N-bioside + H2O
p-nitrophenol + lacto-N-biose
show the reaction diagram
Streptomyces sp. 142
-
-
-
-
pyridylamine-oligosaccharides which have type 1 chains at the nonreducing terminus + H2O
lacto-N-biose + ?
show the reaction diagram
-
-
from N-acetyllactosamine type of triantennary sugar chain with type 1 structure and N-avcetyllactosamine type of biantennary sugar chain is produced
-
pyridylamine-oligosaccharides which have type 1 chains at the nonreducing terminus + H2O
lacto-N-biose + ?
show the reaction diagram
Streptomyces sp., Streptomyces sp. 142
-
no reaction with pyridylamine-oligosaccharides which contain only type 2 chains. No reaction with oligosaccharides in which the non-reducing terminal Gal or the penultimate GlcNAc are replaced by fucose or sialic acid are
-
-
pyridylamine-oligosaccharides which have type 1 chains at the nonreducing terminus + H2O
lacto-N-biose + ?
show the reaction diagram
Streptomyces sp. 142
-
-
from N-acetyllactosamine type of triantennary sugar chain with type 1 structure and N-avcetyllactosamine type of biantennary sugar chain is produced
-
sialyllacto-N-tetraose a + H2O
lactose + Fuc-alpha-(1->2)-Gal-beta-(1->3)[Fuc-alpha-(1->4)]GlcNAc
show the reaction diagram
Bifidobacterium longum, Bifidobacterium longum JCM1217
Neu5Ac-alpha-(2->3)-Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Gal
-
?
lacto-N-tetraose + H2O
lactose + lacto-N-biose
show the reaction diagram
Bifidobacterium longum, Bifidobacterium longum JCM1217
Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc
-
?
additional information
?
-
-
no reaction with lacto-N-neotetraose, lacto-N-triose, sialyl lacto-N-tetraose, lacto-N-fucopentaose I, II, or III, asialo-GM1 tetrasaccharide, poly-N-acetyllactosamine
-
-
-
additional information
?
-
-
no activity on beta-linked p-nitrophenyl-monosaccharides, ganglioside GA1 structure with a beta-linked galacto-N-biose, fucosylated forms of lacto-N-tetraose or lacto-N-neoteraose, the recombinant enzyme shows substrate preference for the unmodified beta-linked lacto-biose I structure
-
-
-
additional information
?
-
-
human milk oligosaccharides are substrates and sole carbon source for Bifidobacterium bifidum colonizing the human intestinal tract. More than 130 types of human milk oligosaccharides have been isolated with the most abundant being lacto-N-tetraose, lacto-N-fucopentaose I, lacto-N-difucohexaose, and 2'-fucosyllactose
-
-
-
additional information
?
-
human milk oligosaccharides are substrates and sole carbon source for Bifidobacterium bifidum colonizing the human intestinal tract. More than 130 types of human milk oligosaccharides have been isolated with the most abundant being lacto-N-tetraose, lacto-N-fucopentaose I, lacto-N-difucohexaose, and 2'-fucosyllactose
-
-
-
additional information
?
-
the native substrate are human milk oligosaccharides. Among the different structures, four molecular species, namely 2'-fucosyllactose, lacto-N-tetraose, lacto-N-fucopentaose I, and lacto-N-difucohexaose I, are most abundantly present and comprise more than 50% of the total oligosaccharides, unless the milk is derived from non-secretor or Lewis-negative subjects
-
-
-
additional information
?
-
the enzyme releases a disaccharide
-
-
-
additional information
?
-
the purified enzyme, which consists of LnbX only, hydrolyzes via a retaining mechanism the GlcNAcbeta-(1->3)-Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I, and sialyllacto-N-tetraose a. Inactive against lacto-N-neotetraose, lacto-N-triose II, lacto-N-fucopentaose II, and sialyllacto-N-tetraose b, substrate specificity, overview
-
-
-
additional information
?
-
Streptomyces sp. 142
-
no reaction with lacto-N-neotetraose, lacto-N-triose, sialyl lacto-N-tetraose, lacto-N-fucopentaose I, II, or III, asialo-GM1 tetrasaccharide, poly-N-acetyllactosamine
-
-
-
additional information
?
-
Bifidobacterium bifidum JCM1254
human milk oligosaccharides are substrates and sole carbon source for Bifidobacterium bifidum colonizing the human intestinal tract. More than 130 types of human milk oligosaccharides have been isolated with the most abundant being lacto-N-tetraose, lacto-N-fucopentaose I, lacto-N-difucohexaose, and 2'-fucosyllactose, the enzyme releases a disaccharide
-
-
-
additional information
?
-
Bifidobacterium longum JCM1217
the native substrate are human milk oligosaccharides. Among the different structures, four molecular species, namely 2'-fucosyllactose, lacto-N-tetraose, lacto-N-fucopentaose I, and lacto-N-difucohexaose I, are most abundantly present and comprise more than 50% of the total oligosaccharides, unless the milk is derived from non-secretor or Lewis-negative subjects, the purified enzyme, which consists of LnbX only, hydrolyzes via a retaining mechanism the GlcNAcbeta-(1->3)-Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I, and sialyllacto-N-tetraose a. Inactive against lacto-N-neotetraose, lacto-N-triose II, lacto-N-fucopentaose II, and sialyllacto-N-tetraose b, substrate specificity, overview
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
D-Gal-(1,3)-beta-GlcNAc-(1,3)-beta-D-Gal-(1,4)-D-Glc + H2O
lactose + D-galactosyl-beta-1,3-N-acetyl-D-glucosamine
show the reaction diagram
-
lacto-N-tetraose, major component of human milk oligosaccharides
lacto-N-biose I
?
D-Gal-(1,3)-beta-GlcNAc-(1,3)-beta-D-Gal-(1,4)-D-Glc + H2O
lactose + D-galactosyl-beta-1,3-N-acetyl-D-glucosamine
show the reaction diagram
Bifidobacterium bifidum, Bifidobacterium bifidum JCM1254
B3TLD6
lacto-N-tetraose, major component of human milk oligosaccharides
lacto-N-biose I
?
lacto-N-fucopentaose I + H2O
lactose + Fuc-alpha-(1->2)-Gal-beta-(1->3)-GlcNAc
show the reaction diagram
Bifidobacterium longum, Bifidobacterium longum JCM1217
A0A024QYS6
Fuc-alpha-(1->2)-Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc
-
?
sialyllacto-N-tetraose a + H2O
lactose + Fuc-alpha-(1->2)-Gal-beta-(1->3)[Fuc-alpha-(1->4)]GlcNAc
show the reaction diagram
Bifidobacterium longum, Bifidobacterium longum JCM1217
A0A024QYS6
Neu5Ac-alpha-(2->3)-Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Gal
-
?
lacto-N-tetraose + H2O
lactose + lacto-N-biose
show the reaction diagram
Bifidobacterium longum, Bifidobacterium longum JCM1217
A0A024QYS6
Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc
-
?
additional information
?
-
-
human milk oligosaccharides are substrates and sole carbon source for Bifidobacterium bifidum colonizing the human intestinal tract. More than 130 types of human milk oligosaccharides have been isolated with the most abundant being lacto-N-tetraose, lacto-N-fucopentaose I, lacto-N-difucohexaose, and 2'-fucosyllactose
-
-
-
additional information
?
-
B3TLD6
human milk oligosaccharides are substrates and sole carbon source for Bifidobacterium bifidum colonizing the human intestinal tract. More than 130 types of human milk oligosaccharides have been isolated with the most abundant being lacto-N-tetraose, lacto-N-fucopentaose I, lacto-N-difucohexaose, and 2'-fucosyllactose
-
-
-
additional information
?
-
A0A024QYS6
the native substrate are human milk oligosaccharides. Among the different structures, four molecular species, namely 2'-fucosyllactose, lacto-N-tetraose, lacto-N-fucopentaose I, and lacto-N-difucohexaose I, are most abundantly present and comprise more than 50% of the total oligosaccharides, unless the milk is derived from non-secretor or Lewis-negative subjects
-
-
-
additional information
?
-
Bifidobacterium bifidum JCM1254
B3TLD6
human milk oligosaccharides are substrates and sole carbon source for Bifidobacterium bifidum colonizing the human intestinal tract. More than 130 types of human milk oligosaccharides have been isolated with the most abundant being lacto-N-tetraose, lacto-N-fucopentaose I, lacto-N-difucohexaose, and 2'-fucosyllactose
-
-
-
additional information
?
-
Bifidobacterium longum JCM1217
A0A024QYS6
the native substrate are human milk oligosaccharides. Among the different structures, four molecular species, namely 2'-fucosyllactose, lacto-N-tetraose, lacto-N-fucopentaose I, and lacto-N-difucohexaose I, are most abundantly present and comprise more than 50% of the total oligosaccharides, unless the milk is derived from non-secretor or Lewis-negative subjects
-
-
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ca2+
required for proper folding of the enzyme LnbX
Mg2+
required for proper folding of the enzyme LnbX
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Gal-beta-(1->3)-GlcNAc-thiazoline
-
-
GalNAc-thiazoline
specific inhibition
-
GlcNAc-thiazoline
specific inhibition
-
guanidine hydrochloride
-
-
N-acetylglucosamine-thiazoline
-
-
-
lacto-N-biose
-
-
additional information
EDTA does not affect the specific activity of the purified enzyme
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
protein LnbY
protein LnbY is absolutely required for proper folding of the enzyme LnbX and its activity. EDTA has no effect on the specific activity of the purified enzyme
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.186
4-nitrophenyl-GalNAc-beta-(1->3)-GlcNAc
recombinant enzyme, pH 5.4, 25C
-
0.119
4-nitrophenyl-lacto-N-bioside
recombinant enzyme, pH 5.4, 25C
-
0.0068
Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc-pyridylamine
-
-
14.6
lacto-N-fucopentaose I
recombinant enzyme, pH 5.4, 25C
0.0068
p-nitrophenyl-Gal-(1,3)-beta-GlcNAc
pH 4.5, 25°C
0.068
p-Nitrophenyl-lacto-N-bioside
-
pH 4.5, 25C
-
0.401
lacto-N-tetraose
recombinant enzyme, pH 5.4, 25C
additional information
additional information
-
-
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
39.4
4-nitrophenyl-GalNAc-beta-(1->3)-GlcNAc
Bifidobacterium longum
A0A024QYS6
recombinant enzyme, pH 5.4, 25C
-
96.1
4-nitrophenyl-lacto-N-bioside
Bifidobacterium longum
A0A024QYS6
recombinant enzyme, pH 5.4, 25C
-
13.5
lacto-N-fucopentaose I
Bifidobacterium longum
A0A024QYS6
recombinant enzyme, pH 5.4, 25C
113
lacto-N-tetraose
Bifidobacterium longum
A0A024QYS6
recombinant enzyme, pH 5.4, 25C
282
lacto-N-tetraose
Bifidobacterium longum
A0A024QYS6
recombinant enzyme, pH 5.4, 25C
89
p-nitrophenyl-Gal-(1,3)-beta-GlcNAc
Bifidobacterium bifidum
B3TLD6
pH 4.5, 25°C
89
p-Nitrophenyl-lacto-N-bioside
Bifidobacterium bifidum JCM 1254
-
pH 4.5, 25C
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
211
4-nitrophenyl-GalNAc-beta-(1->3)-GlcNAc
Bifidobacterium longum
A0A024QYS6
recombinant enzyme, pH 5.4, 25C
202176
806
4-nitrophenyl-lacto-N-bioside
Bifidobacterium longum
A0A024QYS6
recombinant enzyme, pH 5.4, 25C
202175
0.932
lacto-N-fucopentaose I
Bifidobacterium longum
A0A024QYS6
recombinant enzyme, pH 5.4, 25C
8127
282
lacto-N-tetraose
Bifidobacterium longum
A0A024QYS6
recombinant enzyme, pH 5.4, 25C
5569
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.000125
Gal-beta-(1->3)-GlcNAc-thiazoline
pH 4.5, 30C
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37.2
purified recombinant non-tagged enzyme, pH 5.4, 30C
additional information
-
enzyme activity in strains JCM1217, JCM1222, JCM7054, bacteria grown anaerobically in basal lactose medium, 37°C, enzyme assay 3 h - overnight, in 100 mM potassium phosphate buffer pH 7.0 and 4 mM substrate lacto-N-tetraose, HPLC with absorbance measurement of N-acetyl group at 214 nm
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4.5
-
p-nitrophenyl-beta-lacto-N-bioside
4.5
for p-nitrophenyl-Gal-(1,3)-beta-GlcNAc, 50 mM buffers pH 2.5-6.0: citrate-phophate buffer, pH 6.0-8.0: sodium phosphate buffer, pH 8.0-9.0: Tris-HCl buffer
4.5
-
p-nitrophenyl-lacto-N-bioside as substrate
5.5
-
Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc-pyridylamine
6
for D-Gal-(1,3)-beta-GlcNAc-(1,3)-beta-D-Gal-(1,4)-beta-D-Glc-pyridylamine, 50 mM buffers pH 2.5-6.0: citrate-phophate buffer, pH 6.0-8.0: sodium phosphate buffer, pH 8.0-9.0: Tris-HCl buffer
6
-
using pyridylamino oligosaccharides as substrates
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
60
-
p-nitrophenyl-beta-lacto-N-bioside
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
Microbacterium sp. L-101, Streptomyces sp. 142
-
-
-
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
a membrane-anchored extracellular enzyme
-
Manually annotated by BRENDA team
Bifidobacterium bifidum JCM1254
-
a membrane-anchored extracellular enzyme
-
-
Manually annotated by BRENDA team
a membrane-anchored extracellular enzyme
Manually annotated by BRENDA team
Bifidobacterium bifidum JCM1254
-
a membrane-anchored extracellular enzyme
-
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
46000
-
gel filtration
26153
110000
-
apparent molecular mass, SDS-PAGE
695742
112000
-
calculated mass
695742
112000
truncated enzyme without signal peptide and membrane anchor, amino acids 35-1064, SDS-PAGE, and calculated from amino acids
695742
120000
full enzyme, calculated from 350 bp PCR product of 1,112 amino acids
695742
120000
-
predicted mass, 1112 amino acid residues. The deduced amino acid sequence contains a signal peptide and a membrane anchor at the N-terminal and C-terminal ends. This result strongly suggests that LnbB is a membrane-tethered protein with a large extracellular region
695742
356000
recombinant nontagged enzyme, gel filtration
732113
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
?
x * 120000, SDS-PAGE
?
Bifidobacterium bifidum JCM1254
-
x * 120000, SDS-PAGE
-
dimer
2 * 161000, recombinant nontagged enzyme, SDS-PAGE
dimer
Bifidobacterium longum JCM1217
-
2 * 161000, recombinant nontagged enzyme, SDS-PAGE
-
monomer
-
1 * 60000, SDS-PAGE
monomer
Streptomyces sp. 142
-
1 * 60000, SDS-PAGE
-
additional information
three-dimensional structure analysis
additional information
Bifidobacterium bifidum JCM1254
-
three-dimensional structure analysis
-
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified wild-type and selenomethionine-labeled enzymes in complex with Gal-beta-(1->3)-GlcNAc and Gal-beta-(1->3)-GlcNAc-thiazoline, sitting drop vapor diffusion method, mixing of 0.0005 ml of 7 mg/ml protein and 10 mM Gal-beta-(1->3)-GlcNAc with an equal volume of reservoir solution containing 0.2 M potassium sodium tartrate tetrahydrate, 0.1 M sodium citrate, pH 5.6, and 2.0 M ammonium sulfate, X-ray diffraction structure determination and analysis at 1.8 A resolution
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4 - 10
-
4C, 16 h, stable
26153
4 - 7
-
4C, 16 h, stable
26154
4 - 9
-
stable between pH 4 and 9
695742
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
40
88% activity after 30 min incubation
695742
40
-
retains 88% activity after incubation at 40C for 30 min
695742
45
-
30 min, stable
26153
50
-
30 min, 20% loss of activity
26154
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
dilution without Brij58 causes 30-50% inactivation
-
stabilized by 0.05% Brij58, 0.05% Nonidet P-40 or 0.01% bovine serum albumin
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C or -20C, protein concentration 0.01 mg/ml or 0.00005 mg/ml, 0.05% Brij 58, stable for at least 6 months
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni2+-charged HiTrap chelating column chromatography, followed by superdex 200 10/300 GL column chromatography, purity determined by SDS-PAGE with Coomassie brilliant blue R250 staining
recombinant His-tagged wild-type and mutant as well as His-tagged selenomethionine-labeled enzymes from Escherichia coli by nickel affinity and anion exchange chromatography and gel filtration to homogeneity
recombinant protein, as the C-terminally His6-tagged protein
-
recombinant non-tagged enzyme 3.6fold from Escherichia coli by two different steps of anion exchange chromataography and hydrophobic interaction chromatography, followed by gel filtration. Recombinant His-tagged enzyme from Escherichia coli by nickel affinity and anion exchange chromatography, and gel filtration
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Bifidobacterium bifidum grown anaerobically in GAM medium, enzyme gene inserted into plasmid pMW118, expression of truncated form missing signal peptide and membrane anchor (era 35-1064) in Escherichia coli rosetta(DE3) pLacI with plasmid pET23b-lnbB, LB medium, 37°C
C-terminally His6-tagged enzyme
-
gene lnbB, expression of His-tagged wild-type enzyme in Escherichia coli BL21 CodonPlus (DE3)-RIL and of His-tagged selenomethionine-labeled enzyme in Escherichia coli BL21 CodonPlus (DE3)-RIL X
cloned in Escherichia coli Rosetta(DE3)
-
gene lnbX, genomic library construction of Bifidobacterium longum strain JCM1217 in Escherichia coli strain DH5alpha, gene disruption and complementation analysis, recombinant expression of lnbX in non-tagged and C-terminally His-tagged form, coexpression with N-terminall His-tagged LnbY in Escherichia coli. The addition of His-tag to the C-terminus of the protein does not alter the enzymatic properties of LnbX
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D320A
site-directed mutagenesis, the mutant shows significantly reduced kcat, but unaffected Km compared with the wild-type enzyme
D320N
site-directed mutagenesis, the mutant shows significantly reduced kcat, but unaffected Km compared with the wild-type enzyme
Y419F
site-directed mutagenesis, the mutant shows significantly reduced Km and kcat values compared with the wild-type enzyme
D320A
Bifidobacterium bifidum JCM1254
-
site-directed mutagenesis, the mutant shows significantly reduced kcat, but unaffected Km compared with the wild-type enzyme
-
D320N
Bifidobacterium bifidum JCM1254
-
site-directed mutagenesis, the mutant shows significantly reduced kcat, but unaffected Km compared with the wild-type enzyme
-
Y419F
Bifidobacterium bifidum JCM1254
-
site-directed mutagenesis, the mutant shows significantly reduced Km and kcat values compared with the wild-type enzyme
-
H263F
site-directed mutagenesis, the mutant shows significantly reduced kcat compared with the wild-type enzyme
additional information
truncated enzyme without N-terminal signal peptide of 34 amino acids and C-terminal membrane anchor from 1065-1108
H263F
Bifidobacterium bifidum JCM1254
-
site-directed mutagenesis, the mutant shows significantly reduced kcat compared with the wild-type enzyme
-
additional information
Bifidobacterium bifidum JCM1254
-
truncated enzyme without N-terminal signal peptide of 34 amino acids and C-terminal membrane anchor from 1065-1108
-
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
In vitro refolding, overview. Denatured C-terminally His-tagged enzyme LnbX is refolded in the presence and absence of LnbY and metals Ca2and Mg2+. Purified LnbX is denatured in 6 M guanidine HCl. It is diluted 100fold by adding 50 mM HEPES buffer, pH 7.0, containing various concentrations of LnbY and metal ions 0.0-5.0 mM in a total volume of 0.5 ml, followed by immediate dialysis against HEPES buffer containing or not containing metal ions
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
-
synthesis of Galbeta(1-3)GlcNAcbeta(1-3)Galbeta(1-4)Glc, i.e. lacto-neotetraose
synthesis
Microbacterium sp. L-101
-
synthesis of Galbeta(1-3)GlcNAcbeta(1-3)Galbeta(1-4)Glc, i.e. lacto-neotetraose
-
analysis
-
the enzyme may be useful in identifying type 1 structure in glycoconjugates and to distinguish type 1 oligosaccharides from type 2 oligosaccharides
analysis
Streptomyces sp. 142
-
the enzyme may be useful in identifying type 1 structure in glycoconjugates and to distinguish type 1 oligosaccharides from type 2 oligosaccharides
-