Information on EC 3.4.24.68 - tentoxilysin

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The expected taxonomic range for this enzyme is: Clostridium tetani

EC NUMBER
COMMENTARY
3.4.24.68
-
RECOMMENDED NAME
GeneOntology No.
tentoxilysin
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
Hydrolysis of -Gln76-/-Phe- bond in synaptobrevin (also known as neuronal vesicle-associated membrane protein, VAMP)
show the reaction diagram
structure and mechanism
-
Hydrolysis of -Gln76-/-Phe- bond in synaptobrevin (also known as neuronal vesicle-associated membrane protein, VAMP)
show the reaction diagram
structure and mechanism
Clostridium tetani Harvard
-
-
Hydrolysis of -Gln76-/-Phe- bond in synaptobrevin (also known as neuronal vesicle-associated membrane protein, VAMP)
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
hydrolysis of peptide bond
-
-
-
-
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
TeNT
Clostridium tetani Y-IV-3
-
-
-
TeNT-LC protein
-
-
Tentoxylysin
-
-
-
-
tetanospasmin
-
-
Tetanus neurotoxin
-
-
-
-
Tetanus neurotoxin
Clostridium tetani Y-IV-3
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
107231-12-9
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
all toxigenic strains synthesize only one type of neurotoxin
-
-
Manually annotated by BRENDA team
all toxigenic strains synthesize only one type of neurotoxin; Harvard
-
-
Manually annotated by BRENDA team
E 88 (non-sporulating)
-
-
Manually annotated by BRENDA team
strain Y-IV-3
-
-
Manually annotated by BRENDA team
tetanus toxin is encoded on a 74082 kb plasmid containing 61 genes
Q93N27
SwissProt
Manually annotated by BRENDA team
toxigenic strains N3911
-
-
Manually annotated by BRENDA team
Clostridium tetani Harvard
Harvard
-
-
Manually annotated by BRENDA team
Clostridium tetani Y-IV-3
strain Y-IV-3
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
malfunction
-
TeNT cleaves vesicle-associated membrane protein-2, thereby inhibiting neurotransmitter release in the central nervous system to elicit spastic paralysis
physiological function
-
cleavage of synaptobrevin results in inhibition of release of neurotransmitters glycine and gamma-amino butyric acid from inhibitory interneurons causing spastic paralysis
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
rat synaptobrevin 2 + H2O
?
show the reaction diagram
-
catalytic activity of all mutants
-
?
Synaptobrevin + H2O
Hydrolyzed synaptobrevin
show the reaction diagram
-
-
-
-
-
Synaptobrevin + H2O
Hydrolyzed synaptobrevin
show the reaction diagram
-
i.e. VAMP, neuronal vesicle-associated membrane protein, MW 19000, with 2 isoforms in human, chicken, in rat brain: synaptobrevin/VAMP-1 and synaptobrevin/VAMP-2, cleaves at Gln76-Phe77, the same site as botulin neurotoxin B
-
-
-
Synaptobrevin + H2O
Hydrolyzed synaptobrevin
show the reaction diagram
Clostridium tetani Harvard
-
i.e. VAMP, neuronal vesicle-associated membrane protein, MW 19000, with 2 isoforms in human, chicken, in rat brain: synaptobrevin/VAMP-1 and synaptobrevin/VAMP-2, cleaves at Gln76-Phe77, the same site as botulin neurotoxin B
-
-
-
synaptobrevin + H2O
?
show the reaction diagram
-
tetanus neurotoxin receptors are located on the motor neuron plasmalemma at neuromuscular junction, after binding the toxin is internalized inside vesicles of unknown nature and then translocated across the vesicle membrane, i.e. VAMP, neuronal vesicle-associated membrane protein, predominantly exposed to cytosol
-
-
-
synaptobrevin + H2O
?
show the reaction diagram
-
enzyme disables neuroexocytosis apparatus, acts at spinal inhibitory interneurons, blocking release of various neurotransmitters to produce spastic paralysis, clostridial neurotoxins are described as the most toxic substances known
-
-
-
synaptobrevin + H2O
?
show the reaction diagram
-
neurotoxin blocks neurotransmitter release in Aplysia neurons
-
-
-
synaptobrevin + H2O
?
show the reaction diagram
-
TeNT is a zinc metalloprotease, that is produced by anaerobically grown Clostridium tetani in infected tissue, where it binds to ganglioside receptors of peripheral nerves. TeNT is then endocytosed. The A subunit exits from the endosome and undergoes retrograde transport via the nerve axon to the spinal cord of the host, where it specifically cleaves one of the nerve cell SNARE proteins, synaptobrevin, a host nerve cell SNARE protein, purified recombinant His-tagged synaptobrevin expressed in Escherichia coli
-
-
?
synaptobrevin + H2O
?
show the reaction diagram
Clostridium tetani Harvard
-
enzyme disables neuroexocytosis apparatus, acts at spinal inhibitory interneurons, blocking release of various neurotransmitters to produce spastic paralysis, clostridial neurotoxins are described as the most toxic substances known
-
-
-
synaptobrevin-2 + H2O
?
show the reaction diagram
-
i.e. vesicle associated membrane protein-2, VAMP-2, i.e. vesicle associated membrane protein-2, VAMP-2, or Syb2, specific proteolytic cleavage, development of a sensitive in vitro assay method using immobilized recombinant substrate and a highly specific polyclonal antibody against the newly generated C-terminus of the product, overview
-
-
?
vesicle-associated membrane protein VAMP + H2O
?
show the reaction diagram
P04958
-
-
-
?
vesicle-associated membrane protein VAMP + H2O
?
show the reaction diagram
Clostridium tetani, Clostridium tetani Y-IV-3
-
L-chain highly specific for the substrate
-
?
vesicle-associated membrane protein-2 + H2O
?
show the reaction diagram
-
neuronal SNARE protein, i.e. VAMP2
-
-
?
additional information
?
-
-
synaptobrevin-1 (with Val76 instead of Gln76) or short peptides containing the cleavage site of the target protein
-
-
-
additional information
?
-
-
catalytic activity requires reduction of the single interchain disulfide bond of the neurotoxin, no substrates are rat or chicken
-
-
-
additional information
?
-
-
no substrates are rat or chicken
-
-
-
additional information
?
-
-
most powerful known natural toxin
-
?
additional information
?
-
-
most powerful known natural toxin
-
?
additional information
?
-
-
most powerful known natural toxin
-
?
additional information
?
-
-
most powerful known natural toxin, 2 carbohdrate binding sites in the Hcc-domain of tetanus neurotoxin are required for toxicity
-
?
additional information
?
-
-
most powerful known natural toxin, acts by blocking the release of glycine from inhibitory neurons within the spinal cords
-
?
additional information
?
-
-
tetanus neurotoxin is a potent inhibitor of neuroexocytosis. Organization and regulation of the neurotoxin gene. The gene located immediately upstream of the tetanus toxin gene, encodes a positive regulatory protein, TetR
-
-
-
additional information
?
-
-
TeNT high affinity binding to neurons is mediated solely by its gangliosides, both of the W and R pockets are necessary for high affinity binding to neuronal and non-neuronal cells. Gangliosides are functional dual receptors for TeNT, overview
-
-
-
additional information
?
-
-
the conformational changes of the C fragment of tetanus neurotoxin (TeNTHc) resulting from disulfide bond formation reduce the ganglioside-binding activity but do not destroy its immunogenicity as a potent vaccine candidate
-
-
-
additional information
?
-
Clostridium tetani Harvard
-
synaptobrevin-1 (with Val76 instead of Gln76) or short peptides containing the cleavage site of the target protein
-
-
-
additional information
?
-
Clostridium tetani Y-IV-3
-
most powerful known natural toxin
-
?
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
synaptobrevin + H2O
?
show the reaction diagram
-
tetanus neurotoxin receptors are located on the motor neuron plasmalemma at neuromuscular junction, after binding the toxin is internalized inside vesicles of unknown nature and then translocated across the vesicle membrane, i.e. VAMP, neuronal vesicle-associated membrane protein, predominantly exposed to cytosol
-
-
-
synaptobrevin + H2O
?
show the reaction diagram
-
enzyme disables neuroexocytosis apparatus, acts at spinal inhibitory interneurons, blocking release of various neurotransmitters to produce spastic paralysis, clostridial neurotoxins are described as the most toxic substances known
-
-
-
synaptobrevin + H2O
?
show the reaction diagram
-
neurotoxin blocks neurotransmitter release in Aplysia neurons
-
-
-
synaptobrevin + H2O
?
show the reaction diagram
-
TeNT is a zinc metalloprotease, that is produced by anaerobically grown Clostridium tetani in infected tissue, where it binds to ganglioside receptors of peripheral nerves. TeNT is then endocytosed. The A subunit exits from the endosome and undergoes retrograde transport via the nerve axon to the spinal cord of the host, where it specifically cleaves one of the nerve cell SNARE proteins, synaptobrevin
-
-
?
synaptobrevin + H2O
?
show the reaction diagram
Clostridium tetani Harvard
-
enzyme disables neuroexocytosis apparatus, acts at spinal inhibitory interneurons, blocking release of various neurotransmitters to produce spastic paralysis, clostridial neurotoxins are described as the most toxic substances known
-
-
-
synaptobrevin-2 + H2O
?
show the reaction diagram
-
i.e. vesicle associated membrane protein-2, VAMP-2
-
-
?
vesicle-associated membrane protein-2 + H2O
?
show the reaction diagram
-
neuronal SNARE protein, i.e. VAMP2
-
-
?
additional information
?
-
-
most powerful known natural toxin
-
?
additional information
?
-
-
most powerful known natural toxin
-
?
additional information
?
-
-
most powerful known natural toxin
-
?
additional information
?
-
-
most powerful known natural toxin, 2 carbohdrate binding sites in the Hcc-domain of tetanus neurotoxin are required for toxicity
-
?
additional information
?
-
-
most powerful known natural toxin, acts by blocking the release of glycine from inhibitory neurons within the spinal cords
-
?
additional information
?
-
-
tetanus neurotoxin is a potent inhibitor of neuroexocytosis. Organization and regulation of the neurotoxin gene. The gene located immediately upstream of the tetanus toxin gene, encodes a positive regulatory protein, TetR
-
-
-
additional information
?
-
-
TeNT high affinity binding to neurons is mediated solely by its gangliosides, both of the W and R pockets are necessary for high affinity binding to neuronal and non-neuronal cells. Gangliosides are functional dual receptors for TeNT, overview
-
-
-
additional information
?
-
Clostridium tetani Y-IV-3
-
most powerful known natural toxin
-
?
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Cobalt
-
zinc-dependent endoproteinase, can replace zinc
Nickel
-
zinc-dependent endoproteinase, can replace zinc
Zinc
-
toxin surface topography of His-residues; zinc-dependent endoproteinase
Zinc
-
zinc-dependent endoproteinase
Zinc
-
1 atom zinc per molecule toxin, zinc-binding motif: His-Glu-X-X-His, nickel or cobalt can replace zinc; zinc-dependent endoproteinase
Zinc
-
L-chain: form of zinc-endopeptidase, 0.8-1 gatom zinc/mol toxin, bound to light or L-chain; zinc-dependent endoproteinase
Zn2+
-
zinc endopeptidase, zinc active site detected by X-ray analysis
Zn2+
-
zinc-dependent endopeptidase
Zn2+
-
zinc endopeptidase
Zn2+
P04958
crystal structure provides insight into the role of the zinc ion
Zn2+
-
dependent on
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
Ala-Ser-Gln-Phe-Glu-Thr-Ser
-
synthetic peptide containing cleavage site of synaptobrevin, inhibits toxin action on buccal ganglion of Aplysia californica
NaOCl
-
inactivation
Gln-Phe-Glu-Thr
-
synthetic peptide containing cleavage site of synaptobrevin, inhibits toxin action on buccal ganglion of Aplysia californica
additional information
-
enzyme inhibition by a cleavage site-specific antibody, overview
-
additional information
-
design, random selection, and recombinant production from phagemid in Escherichia coli of human single chain antibody fragments that inhibit tetanus toxin binding to retinoic acid pulsed human neuroblastoma cells and TeNT protease activity completely, overview
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
Proteases
-
activation by rapid cleavage within an exposed loop of the single inactive MW 150000 polypeptide chain and generation of active di-chain neurotoxin; bacterial; or tissue proteases
-
Proteases
-
activation by rapid cleavage within an exposed loop of the single inactive MW 150000 polypeptide chain and generation of active di-chain neurotoxin; bacterial
-
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
-
in neurotoxin-injected Aplysia neurons 4-10 molecules of L-chains are sufficient to cause blockade of neurotransmitter release with a t1/2 of 20-40 min at 20C
additional information
-
-
quantitative immunoaffinity assay method development and evaluation, overview
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
7
-
-
assay at
7.4
-
-
assay at
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
Clostridium tetani Harvard
-
-
-
Manually annotated by BRENDA team
additional information
-
effect of medium composition on the production of tetanus toxin. The highest final average yield of tetanus toxin is obtained at 9.7 mg/ml starting level of glucose and 43.5 g/l N-T Case TT as carbon and nitrogen source
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
accumulates until bacterial lysis
Manually annotated by BRENDA team
Clostridium tetani Harvard
-
accumulates until bacterial lysis
-
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
Clostridium tetani (strain Massachusetts / E88)
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
150000
-
-
single-chain protein, cleaved by proteases into a 100 kDa heavy chain HC and a 50 kDa light chain LC, SELDI-TOF MS
150000
-
-
activated by proteolytic cleavage with formation of a 50 kDa L-chain and a 100 kDa H-chain
150700
-
-
Clostridium tetani, calculated from amino acid sequence
additional information
-
-
amino acid sequence homologies between tetanus toxin TeNT and botulinum toxins BoNT/A, B and E
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
dimer
-
1 * 100000, C-terminal heavy chain, + 1 * 50000, N-terminal light chain
additional information
-
held together by a single disulfide bond and non-covalent forces; the enzyme consists of a heavy (H) chain and a light (L) chain
additional information
-
MW 52288 (L-chain) and MW 98300 (H-chain), calculated from amino acid sequence; the enzyme consists of a heavy (H) chain and a light (L) chain
additional information
-
TeNT is an A-B toxin. It consits of a heavy chain containing cellular receptor binding domain and a light chain with zinc metalloprotease activity
additional information
-
the TeNT heavy chain contains two functional domains: a translocation domain and a C-terminal receptor binding domain. C-terminal heavy and N-terminal light chains are linked through a single disulfide bond, overview
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
glycolipoprotein
-
gangliosides are bound to the C-terminal receptor-binding domain via two carbohydrate-binding sites, termed the lactose-binding site or the W pocket, and the sialic acid-binding site or the R pocket, GM1a bound to the W pocket, and GD3 bound to the R pocket
proteolytic modification
-
TeNT is produced as a 150-kDa protein that is cleaved to a di-chain protein, comprising an N-terminal light chain and a C-terminal heavy chain domain linked through a single disulfide bond
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
crystals obtained with the hanging drop method
-
purified C-terminal receptor binding domain with bound ganglioside GT2 in presence and absence of lactose, 10 mg/ml protein in 20 mM Tris-HCl buffer, pH 7.9, containing 100 mM NaCl are mixed with the carbohydrate moiety of GT2 at 1:8 molar ratio, vapor diffusion hanging drop method, 0.002 ml of protein-ligand solution are mixed with 0.002 ml of well solution containing 100 mM bis(trispropane) buffer, pH 7.0, 25% polyethylene glycol 2000 and 300 mM ammonium sulfate, equilibration against 0.5 ml well solution at 19C, X-ray diffraction structure determination and analysis at 2.0-2.1 A resolution, molecular replacement method
-
sitting drop vapor diffusion method. The structure provides insight into the active site, the importance of the nucleophilic water and the role of the zinc ion
P04958
tetanus neurotoxin C fragment, vapor-diffusion method, hanging drops and sitting drops, thick rod-shaped crystals, space group P2(1)2(1)2(1), unit cell dimensions a : 67.4 A, b : 79.7 A, c : 91.1 A
-
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
50
60
-
Tm-value 58.4C
OXIDATION STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
extremely sensitive to oxidants
-
31426
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
-80C, in 10 mM HEPES buffer, pH 7.2, 50 mM NaCl, after freezing in liquid N2, stable
-
4C, both native and recombinant TeNT L-chains show significant decreases after 3-4 days of storage
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
His-tagged teTN-LC protein
P04958
single-chain, two-chain and L-chain form; very toxic! Booster injection of tetanus toxoid before starting research with tetanus toxin advisable, human anti-tetanus neurotoxin antibodies available
-
TeNT Hc fragment mutants
-
tetanus neurotoxin light chain
-
wild-type and mutated recombinant Hc-fragments
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
Clostridium tetani
-
Clostridium tetani; expressed in Escherichia coli JM101 using three different plasmid vectors
-
expressed in Escherichia coli
-
tetanus neurotoxin light chain, expressed in Escherichia coli
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
D1222L
-
lactose-binding site mutant, mutation generated by PCR
D1309A
-
site-directed mutagenesis
D1309N
-
site-directed mutagenesis
E1310A
-
site-directed mutagenesis
E1310Q
-
site-directed mutagenesis
F1305A
-
site-directed mutagenesis
G1215F
-
sialic acid binding site mutant, mutation generated by PCR
G1300F
-
lactose-binding site mutant, mutation generated by PCR
H1271A
-
lactose-binding site mutant, mutation generated by PCR
H1271W
-
lactose-binding site mutant, mutation generated by PCR
H1293A
-
lactose-binding site mutant, mutation generated by PCR
K1295A
-
site-directed mutagenesis
N1219I
-
lactose-binding site mutant, mutation generated by PCR
N1220I
-
lactose-binding site mutant, mutation generated by PCR
R1168A
-
site-directed mutagenesis
R1168K
-
site-directed mutagenesis
R1226F
-
sialic acid binding site mutant, mutation generated by PCR
R1226L
-
sialic acid binding site mutant, mutation generated by PCR
S1287A
-
lactose-binding site mutant, mutation generated by PCR
W1289G
-
lactose-binding site mutant, mutation generated by PCR
W1289L
-
lactose-binding site mutant, mutation generated by PCR
W1303A
-
site-directed mutagenesis
Y1170A
-
site-directed mutagenesis
Y1290A
-
lactose-binding site mutant, mutation generated by PCR
Y1290F
-
site-directed mutagenesis
Y1290F
-
lactose-binding site mutant, mutation generated by PCR
Y1290K
-
site-directed mutagenesis
Y1290S
-
site-directed mutagenesis
Y1292K
-
site-directed mutagenesis
K1297A
-
site-directed mutagenesis
additional information
-
construction of mutated forms of HCR/T that lack one or both carbohydrate-binding pocket, loss of gangliosides binding ability leads to loss of neuron binding ability of the toxin, both of the W and R pockets are necessary for high affinity binding to neuronal and non-neuronal cells, overview
APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
medicine
-
sole causal agent of the pathological condition known as tetanus
medicine
-
sole cause of the devastating disease tetanus
medicine
-
causative agent of the disease tetanus
medicine
-
very powerful neurotoxin, agent responsible for all clinical symptoms of tetanus
medicine
Clostridium tetani Y-IV-3
-
very powerful neurotoxin, agent responsible for all clinical symptoms of tetanus
-