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Information on EC 3.2.1.140 - lacto-N-biosidase and Organism(s) Bifidobacterium bifidum and UniProt Accession B3TLD6
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3.2.1.140 lacto-N-biosidaseIUBMB Comments
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
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Word Map
- 3.2.1.140
- oligosaccharides
- bifidum
- lacto-n-tetraose
- milk
- infant
- glycoside
-
gal
- glcnac
- lactose
- lacto-n-fucopentaose
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triantennary
- lacto-n-triose
-
gut-associated
- lacto-n-neotetraose
-
bifidobacterial
- tetrasaccharide
-
transglycosylation
- glycosidases
- n-acetyllactosamine
-
breast-fed
-
galactosyl
- 2-aminopyridine
-
commensal
-
sialyl
- microbiota
-
subsite
- analysis
- synthesis
The taxonomic range for the selected organisms is: Bifidobacterium bifidum
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
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Synonyms
lacto-n-biosidase, lnbase, bllj_1505, bllj_1506, bblnbase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
lacto-N-biosidase
lacto-N-biosidase (Streptomyces strain 142)
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lacto-N-biosidase
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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
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
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
the enzyme performs a two-step catalytic mechanism involving substrate-assisted catalysis that forms a transient oxazoline or oxazolinium ion intermediate, mechanism overview
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hydrolysis of O-glycosyl bond
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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
CAS REGISTRY NUMBER
COMMENTARY
146359-52-6
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183511-11-7
lacto-N-biosidase (Streptomyces strain 142)
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-methylumbelliferyl 2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl)-D-glucopyranoside + H2O
4-methylumbelliferol + 2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl)-D-glucopyranose
-
-
-
?
4-nitrophenyl-beta-lacto-N-bioside + H2O
4-nitrophenol + lacto-N-biose
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?
4-nitrophenyl-Gal-beta-(1->3)-GalNAc + H2O
4-nitrophenol + Gal-beta-(1->3)-GalNAc
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-
-
?
D-Gal-(1,3)-beta-GlcNAc-(1,3)-beta-D-Gal-(1,4)-beta-D-Glc-pyridylamine + H2O
?
lacto-N-tetraose-pyridylamine, no activity on alpha-linked disaccharides, beta-linked p-nitrophenyl monosaccharides such as 4-nitrophenyl beta-D-N-acetylglucosamine and 4-nitrophenyl beta-D-N-acetyl-D-galactosamine, 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
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-
?
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
lacto-N-tetraose, major component of human milk oligosaccharides
lacto-N-biose I
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?
Gal-beta-(1->3)-GalNAc-beta-(1->3)-Gal-alpha-(1->4)-Gal-beta-(1->4)-Glc + H2O
Gal-beta-(1->3)-GalNAc + Gal-alpha-(1->4)-Gal-beta-(1->4)-Glc
i.e. Gb5
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-
?
Gal-beta-(1->3)-GalNAc-beta-(1->4)-Gal-beta-(1->4)-Glc + H2O
Gal-beta-(1->3)-GlcNAc + Gal-beta-(1->4)-Glc
i.e. GA1
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-
?
Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc + H2O
Gal-beta-(1->3)-GlcNAc + Gal-beta-(1->4)-Glc
i.e. LNT
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-
?
p-nitrophenyl-Gal-(1,3)-beta-GalNAc + H2O
?
30% enzyme activity compared to p-nitrophenyl-Gal-(1,3)-beta-GlcNAc
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?
p-nitrophenyl-Gal-(1,3)-beta-GlcNAc + H2O
?
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
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?
2'-fucosyllactose + H2O
lactose + fucose
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Fuc-alpha-(1->2)-Gal-beta-(1->4)-Glc
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?
lacto-N-difucohexaose + H2O
lactose + Fuc-alpha-(1->2)-Gal-beta-(1->3)[Fuc-alpha-(1->4)]GlcNAc
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Fuc-alpha-(1->2)Gal-beta-(1->3)[Fuc-alpha-(1->4)]GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc
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?
lacto-N-fucopentaose I + H2O
lactose + Fuc-alpha-(1->2)-Gal-beta-(1->3)-GlcNAc
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Fuc-alpha-(1->2)-Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc
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?
lacto-N-tetraose + H2O
lactose + lacto-N-biose
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Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc
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?
lacto-N-tetraose + H2O
lacto-N-biose + lactose
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?
additional information
?
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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
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-
?
additional information
?
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-
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
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-
?
additional information
?
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the enzyme releases a disaccharide
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?
additional information
?
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the enzyme releases a disaccharide
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?
additional information
?
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LnbB, a member of the glycoside hydrolase family 20 isolated from Bifidobacterium bifidum, releases Galbeta1-3GalNAc (GNB) from Gb5 and GA1 oligosaccharides. Substrate specificity, overview. Glycan-scavenging activity of LnbB, i.e. the activity of the enzyme towards the oligosaccharides of globo- and ganglio-series sphingolipids. Activity of GH20 LnbB towards Gb5 oligosaccharide is significantly low. No activity with Fuc-alpha-(1->2)-Gal-beta-(1->3)-GalNAc-beta-(1->3)-Gal-alpha-(1->4)-Gal-beta-(1->4)-Glc (Globo H) and Fuc-alpha-(1->2)Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc (LNFP I)
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?
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
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?
NATURAL SUBSTRATE
NATURAL 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
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
lacto-N-tetraose, major component of human milk oligosaccharides
lacto-N-biose I
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?
lacto-N-tetraose + H2O
lacto-N-biose + lactose
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-
-
?
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
?
-
-
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
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-
?
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl)-D-gluconohydroxyimino-1,5-lactone
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2-acetamido-3-O-(beta-D-galactopyranosyl)-1,5-imino-1,2,5-trideoxy-D-glucitol
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6-acetamido-6-deoxy-7-O-(beta-D-galactopyranosyl)castanospermine
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O-(2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl)-D-glucopyranosylidene)amino N-phenyl carbamate
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additional information
small molecule inhibitors docking to enzyme LnbB, compound synthesis, structure analysis and inhibition mode, NMR spectrometrci analysis, overview
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0088
Gal-beta-(1->3)-GalNAc-beta-(1->3)-Gal-alpha-(1->4)-Gal-beta-(1->4)-Glc
pH 6.0, 30°C
67
Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc
pH 6.0, 25°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.29
Gal-beta-(1->3)-GalNAc-beta-(1->3)-Gal-alpha-(1->4)-Gal-beta-(1->4)-Glc
pH 6.0, 30°C
42
Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc
pH 6.0, 25°C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
39.96
Gal-beta-(1->3)-GalNAc-beta-(1->3)-Gal-alpha-(1->4)-Gal-beta-(1->4)-Glc
pH 6.0, 30°C
0.63
Gal-beta-(1->3)-GlcNAc-beta-(1->3)-Gal-beta-(1->4)-Glc
pH 6.0, 25°C
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.000125
1,3-bis[4-(5,6-dimethyl-1H-benzimidazol-2-yl)phenyl]urea
pH 4.5, 37°C
0.0077
2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl)-D-gluconohydroxyimino-1,5-lactone
pH 4.5, 37°C
0.00088
2-acetamido-3-O-(beta-D-galactopyranosyl)-1,5-imino-1,2,5-trideoxy-D-glucitol
pH 4.5, 37°C
0.00052
6-acetamido-6-deoxy-7-O-(beta-D-galactopyranosyl)castanospermine
pH 4.5, 37°C
0.052
8-acetamido-8-deoxy-2-O-(beta-D-galactopyranosyl)australine
pH 4.5, 37°C
0.000091
O-(2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl)-D-glucopyranosylidene)amino N-phenyl carbamate
pH 4.5, 37°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4.5
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
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
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
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
additional information
evolution
the enzyme belongs to the glycoside hydrolase family 20, GH20
evolution
LnbB is a member of the glycoside hydrolase family 20, GH20
evolution
the enzyme belongs to the glycosyl hydrolase family 20, GH20
evolution
the enzyme belongs to the glycosyl hydrolase family 20, GH20. Domain organization of GH20 beta-N-acetylhexosaminidases, overview
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
lacto-N-biosidase (LNBase), a beta-N-acetyl-hexosaminidase that liberates lacto-N-biose (LNB) from human milk oligosaccharides (HMOs), is important to the LNB pathway
physiological function
lacto-N-biosidase is a key enzyme that degrades lacto-N-tetraose, a main component of human milk oligosaccharides (HMOs, the third-most abundant solid component in human milk), into lacto-N-biose I and lactose. . Enzymatic release of beta-linked GNB from natural substrates, these unique activities may play a role in modulating the microbial composition in the gut ecosystem
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
-
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
the large Bifidobacterium bifidum lacto-N-biosidase and its truncated mutants are used as model proteins to evaluate the minimal functional unit due to its interest and structural complexity. Structure-function analysis of the wild-type in comparison to different truncated enzyme mutants, and comparisons of enzyme structure models, overview. The lacto-N-biosidase requires GH20b and the lectin-like domain at the N- and C-termini of the catalytic GH20 domain to be fully soluble and functional. The lectin domain provides this remote element to the active site. Restoration of activity of the inactive GH20beta-GH20-alpha construct (model A architecture) by a complementation assay with the lectin-like domain
additional information
-
the large Bifidobacterium bifidum lacto-N-biosidase and its truncated mutants are used as model proteins to evaluate the minimal functional unit due to its interest and structural complexity. Structure-function analysis of the wild-type in comparison to different truncated enzyme mutants, and comparisons of enzyme structure models, overview. The lacto-N-biosidase requires GH20b and the lectin-like domain at the N- and C-termini of the catalytic GH20 domain to be fully soluble and functional. The lectin domain provides this remote element to the active site. Restoration of activity of the inactive GH20beta-GH20-alpha construct (model A architecture) by a complementation assay with the lectin-like domain
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
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
112000
truncated enzyme without signal peptide and membrane anchor, amino acids 35-1064, SDS-PAGE, and calculated from amino acids
120000
full enzyme, calculated from 350 bp PCR product of 1,112 amino acids
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
three-dimensional enzyme structure analysis, overview
additional information
-
three-dimensional enzyme structure analysis, overview
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified enzyme in complex with inhibitors 2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl)-D-gluconohydroxyimino-1,5-lactone, 2-acetamido-3-O-(beta-D-galactopyranosyl)-1,5-imino-1,2,5-trideoxy-D-glucitol, 6-acetamido-6-deoxy-7-O-(beta-D-galactopyranosyl)castanospermine, and 8-acetamido-8-deoxy-2-O-(beta-D-galactopyranosyl)australine, sitting drop vapor diffusion method, mixing of 0.001 ml of 10 mg/ml protein solution containing 0.1 mM ligand, with an equal volume of a reservoir solution containing 0.2 M potassium sodium tartrate tetrahydrate, 0.1 M sodium citrate, pH 5.6, and 2.0 M ammonium sulfate, 20°C, X-ray diffraction structure determination and analysis at 1.6-2.2 A resolution, modeling
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
PROTEIN VARIANTS
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
H263F
site-directed mutagenesis, the mutant shows significantly reduced kcat 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
additional information
additional information
truncated enzyme without N-terminal signal peptide of 34 amino acids and C-terminal membrane anchor from 1065-1108
additional information
-
truncated enzyme without N-terminal signal peptide of 34 amino acids and C-terminal membrane anchor from 1065-1108
additional information
generation of different truncated forms of the enzyme, structure-function analysis in comparison to the wild-type, overview. The large Bifidobacterium bifidum lacto-N-biosidase and its truncated mutants are used as a model proteins to evaluate the minimal functional unit due to its interest and structural complexity
additional information
-
generation of different truncated forms of the enzyme, structure-function analysis in comparison to the wild-type, overview. The large Bifidobacterium bifidum lacto-N-biosidase and its truncated mutants are used as a model proteins to evaluate the minimal functional unit due to its interest and structural complexity
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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 enzyme from Escherichia coli strain BL21(DE3)
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) Star by nickel affinity chromatography, gel filtration, and ultrafiltration
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
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
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
recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3) Star
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Wada, J.; Ando, T.; Kiyohara, M.; Ashida, H.; Kitaoka, M.; Yamaguchi, M.; Kumagai, H.; Katayama, T.; Yamamoto, K.
Bifidobacterium bifidum lacto-N-biosidase, a critical enzyme for the degradation of human milk oligosaccharides with a type 1 structure
Appl. Environ. Microbiol.
74
3996-4004
2008
Bifidobacterium bifidum (B3TLD6), Bifidobacterium bifidum, Bifidobacterium bifidum JCM 1254, Bifidobacterium longum, no activity in Bifidobacterium breve, no activity in Bifidobacterium catenulatum
Hattie, M.; Debowski, A.W.; Stubbs, K.A.
Development of tools to study lacto-N-biosidase: an important enzyme involved in the breakdown of human milk oligosaccharides
ChemBioChem
13
1128-1131
2012
Bifidobacterium bifidum
Ito, T.; Katayama, T.; Hattie, M.; Sakurama, H.; Wada, J.; Suzuki, R.; Ashida, H.; Wakagi, T.; Yamamoto, K.; Stubbs, K.A.; Fushinobu, S.
Crystal structures of a glycoside hydrolase family 20 lacto-N-biosidase from Bifidobacterium bifidum
J. Biol. Chem.
288
11795-11806
2013
Bifidobacterium bifidum (B3TLD6), Bifidobacterium bifidum
Gotoh, A.; Katoh, T.; Sugiyama, Y.; Kurihara, S.; Honda, Y.; Sakurama, H.; Kambe, T.; Ashida, H.; Kitaoka, M.; Yamamoto, K.; Katayama, T.
Novel substrate specificities of two lacto-N-biosidases towards beta-linked galacto-N-biose-containing oligosaccharides of globo H, Gb5, and GA1
Carbohydr. Res.
408
18-24
2015
Bifidobacterium bifidum (B3TLD6), Bifidobacterium longum subsp. Longum (A0A024QYS6)
Hattie, M.; Ito, T.; Debowski, A.W.; Arakawa, T.; Katayama, T.; Yamamoto, K.; Fushinobu, S.; Stubbs, K.A.
Gaining insight into the catalysis by GH20 lacto-N-biosidase using small molecule inhibitors and structural analysis
Chem. Commun. (Camb.)
51
15008-15011
2015
Bifidobacterium bifidum (B3TLD6)
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