| IED ID | IndEnz0002014607 |
| Enzyme Type ID | protease014607 |
| Protein Name |
Botulinum neurotoxin type B BoNT/B Bontoxilysin-B Cleaved into: Botulinum neurotoxin B light chain LC EC 3.4.24.69 ; Botulinum neurotoxin B heavy chain HC |
| Gene Name | botB |
| Organism | Clostridium botulinum |
| Taxonomic Lineage | cellular organisms Bacteria Terrabacteria group Firmicutes Clostridia Eubacteriales Clostridiaceae Clostridium Clostridium botulinum |
| Enzyme Sequence | MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFNKSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLGDRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNHFASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLYGIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETNIAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKAINKQAYEEISKEHLAVYKIQMCKSVKAPGICIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSNYIENDFPINELILDTDLISKIELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQYLYSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVNDFVIEANKSNTMDKIADISLIVPYIGLALNVGNETAKGNFENAFEIAGASILLEFIPELLIPVVGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMYKALNYQAQALEEIIKYRYNIYSEKEKSNINIDFNDINSKLNEGINQAIDNINNFINGCSVSYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENKLYLIGSAEYEKSKVNKYLKTIMPFDLSIYTNDTILIEMFNKYNSEILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKLFLAPISDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE |
| Enzyme Length | 1291 |
| Uniprot Accession Number | P10844 |
| Absorption | |
| Active Site | ACT_SITE 231; /evidence=ECO:0000255|PROSITE-ProRule:PRU10095 |
| Activity Regulation | ACTIVITY REGULATION: Proteolysis inhibited by EDTA and captopril, and by peptides that encompass the VAMP2 cleavage site (Ala-Ser-Gln-Phe-Glu-Thr-Ser and Gln-Phe-Glu-Thr) (PubMed:1331807). Translocation of whole toxin into neurons is inhibited by toosendanin (PubMed:21925111). {ECO:0000269|PubMed:1331807, ECO:0000269|PubMed:21925111}. |
| Binding Site | BINDING 1025; /note=Host ganglioside GT1b binding; via amide nitrogen; /evidence=ECO:0007744|PDB:4KBB |
| Calcium Binding | |
| catalytic Activity | CATALYTIC ACTIVITY: Reaction=Limited hydrolysis of proteins of the neuroexocytosis apparatus, synaptobrevins, SNAP25 or syntaxin. No detected action on small molecule substrates.; EC=3.4.24.69; Evidence={ECO:0000269|PubMed:1331807}; |
| DNA Binding | |
| EC Number | 3.4.24.69 |
| Enzyme Function | FUNCTION: [Botulinum neurotoxin type B]: Botulinum toxin causes flaccid paralysis by inhibiting neurotransmitter (acetylcholine) release from the presynaptic membranes of nerve terminals of the eukaryotic host skeletal and autonomic nervous system, with frequent heart or respiratory failure. Precursor of botulinum neurotoxin B which has 2 coreceptors; complex polysialylated gangliosides found on neural tissue and specific membrane-anchored proteins found in synaptic vesicles (PubMed:17185412, PubMed:17167421, PubMed:17167418, PubMed:23807078). Receptor proteins are exposed on host presynaptic cell membrane during neurotransmitter release, when the toxin heavy chain (HC) binds to them (PubMed:14504267). Upon synaptic vesicle recycling the toxin is taken up via the endocytic pathway (PubMed:14504267). When the pH of the toxin-containing endosome drops a structural rearrangement occurs so that the N-terminus of the HC forms pores that allows the light chain (LC) to translocate into the cytosol (PubMed:3856850). Once in the cytosol the disulfide bond linking the 2 subunits is reduced and LC cleaves its target protein on synaptic vesicles, preventing their fusion with the cytoplasmic membrane and thus neurotransmitter release. Binds to host peripheral neuronal presynaptic membranes via synaptotagmins 1 and 2 (SYT1 and SYT2) (PubMed:8144634, PubMed:14504267). Toxin binds to the membrane proximal extra-cytoplasmic region of host SYT1 and SYT2 that is transiently exposed outside of cells during exocytosis; exogenous gangliosides enhance binding and subsequent uptake of toxin into host cells (PubMed:14504267, PubMed:15123599). Toxin uptake into neural cells requires stimulation (incubation with K(+) to stimulate SYT protein receptor exposure); subsequently the toxin colocalizes with its receptor in host cells with a concomitant decrease in target protein (synaptobrevin-2/VAMP2) immunoreactivity (PubMed:14504267). Toxin uptake can be blocked by the appropriate synaptotagmin protein fragments and gangliosides in cell culture and in mice (PubMed:14504267, PubMed:15123599). BoNT/B is a 'coincidence detector'; it requires simultaneous binding to coreceptor GT1b and low pH to transform into a membrane-bound, oligomeric channel (PubMed:21925111, PubMed:22720883). Whole toxin only has protease activity after reduction which releases LC (PubMed:1331807, PubMed:7803399). {ECO:0000269|PubMed:1331807, ECO:0000269|PubMed:14504267, ECO:0000269|PubMed:15123599, ECO:0000269|PubMed:17167418, ECO:0000269|PubMed:17167421, ECO:0000269|PubMed:17185412, ECO:0000269|PubMed:21925111, ECO:0000269|PubMed:22720883, ECO:0000269|PubMed:23807078, ECO:0000269|PubMed:3856850, ECO:0000269|PubMed:7803399, ECO:0000269|PubMed:8144634}.; FUNCTION: [Botulinum neurotoxin B light chain]: Has proteolytic activity (PubMed:1331807). After translocation into the eukaryotic host cytosol, inhibits neurotransmitter release by acting as a zinc endopeptidase that cleaves the '76-Gln-|-Phe-77' bond of synaptobrevin-2/VAMP2, blocking neurotransmitter release (PubMed:1331807, PubMed:7803399). In vitro the LC only has protease activity after reduction (PubMed:1331807, PubMed:7803399). {ECO:0000269|PubMed:1331807, ECO:0000305|PubMed:7803399}.; FUNCTION: [Botulinum neurotoxin B heavy chain]: Responsible for host epithelial cell transcytosis, host nerve cell targeting and translocation of light chain (LC) into host cytosol. Composed of 3 subdomains; the translocation domain (TD), and N-terminus and C-terminus of the receptor-binding domain (RBD). The N-terminus of the TD wraps an extended belt around the perimeter of the LC; it does not seem to protect the active site, but might prevent premature LC dissociation from the translocation channel and protect toxin prior to translocation (PubMed:10932256, PubMed:17167418). Has 2 coreceptors; complex gangliosides found primarily on neural tissue and host synaptotagmin-1 and -2 (SYT1 and SYT2) which bind simultaneously to adjacent but separate sites at the tip of the HC (PubMed:8144634, PubMed:17185412, PubMed:17167421, PubMed:17167418, PubMed:23807078). HC alone partially prevents uptake of whole toxin by neural cells, and delays paralysis onset by 160% (PubMed:10413679). Binding probably positions the TD for integration into the synaptic vesicle membrane (PubMed:17167418, PubMed:23807078). The HC forms channels at low pH that mediate transport of the light chain (LC) from the endocytic vesicle to the cytosol (PubMed:3856850). Binds gangliosides GD1b and GT1b (PubMed:10413679, PubMed:14731268). Gangliosides are not only a coreceptor, but also required for uptake into nerve cells (PubMed:21925111, PubMed:17167418). HC alone binds to host receptor proteins SYT1 and SYT2 (PubMed:14504267, PubMed:15123599, PubMed:17185412, PubMed:19650874). Interaction with SYT1 protein does not require SYT1 glycosylation (PubMed:19476346). The HC C-terminus (approximately residues 1079-1291) interacts with host SYT2 (PubMed:15123599, PubMed:17167421, PubMed:17167418, PubMed:23807078). Has higher affinity for SYT2 than SYT1 (PubMed:17185412, PubMed:17167421). Significantly decreases uptake and toxicity of whole BoNT/B and BoNT/G (PubMed:19650874). {ECO:0000269|PubMed:10413679, ECO:0000269|PubMed:10932256, ECO:0000269|PubMed:14504267, ECO:0000269|PubMed:14731268, ECO:0000269|PubMed:15123599, ECO:0000269|PubMed:17167418, ECO:0000269|PubMed:17167421, ECO:0000269|PubMed:17185412, ECO:0000269|PubMed:19650874, ECO:0000269|PubMed:23807078, ECO:0000269|PubMed:8144634, ECO:0000305|PubMed:21925111, ECO:0000305|PubMed:3856850}. |
| Temperature Dependency | |
| PH Dependency | |
| Pathway | |
| nucleotide Binding | |
| Features | Active site (1); Beta strand (78); Binding site (1); Chain (3); Disulfide bond (1); Helix (44); Initiator methionine (1); Metal binding (3); Motif (1); Mutagenesis (17); Region (6); Sequence conflict (4); Turn (8) |
| Keywords | 3D-structure;Direct protein sequencing;Disulfide bond;Host cell junction;Host cell membrane;Host cytoplasm;Host cytoplasmic vesicle;Host membrane;Host synapse;Hydrolase;Lipid-binding;Membrane;Metal-binding;Metalloprotease;Neurotoxin;Pharmaceutical;Protease;Secreted;Toxin;Transmembrane;Virulence;Zinc |
| Interact With | P29101; P46097 |
| Induction | |
| Subcellular Location | SUBCELLULAR LOCATION: [Botulinum neurotoxin type B]: Secreted {ECO:0000269|PubMed:3139097}. Host cell junction, host synapse, host presynaptic cell membrane {ECO:0000269|PubMed:14504267}. Note=Colocalizes with its SYT1 receptor, probably in synaptic vesicles (PubMed:14504267). At pH 4.4 in the presence of ganglioside GT1b becomes a membrane-associated hydrophobic protein (PubMed:21925111). {ECO:0000269|PubMed:14504267, ECO:0000269|PubMed:21925111}.; SUBCELLULAR LOCATION: [Botulinum neurotoxin B light chain]: Secreted {ECO:0000269|PubMed:3139097}. Host cytoplasm, host cytosol {ECO:0000305|PubMed:1331807, ECO:0000305|PubMed:8144634}.; SUBCELLULAR LOCATION: [Botulinum neurotoxin B heavy chain]: Secreted {ECO:0000269|PubMed:3139097}. Host cell junction, host synapse, host presynaptic cell membrane {ECO:0000269|PubMed:14504267}. Host cytoplasmic vesicle, host secretory vesicle, host synaptic vesicle membrane {ECO:0000305|PubMed:21925111}; Multi-pass membrane protein {ECO:0000305}. |
| Modified Residue | |
| Post Translational Modification | |
| Signal Peptide | |
| Structure 3D | X-ray crystallography (31) |
| Cross Reference PDB | 1EPW; 1F31; 1F82; 1G9A; 1G9B; 1G9C; 1G9D; 1I1E; 1S0B; 1S0C; 1S0D; 1S0E; 1S0F; 1S0G; 1Z0H; 2ETF; 2NM1; 2NP0; 2XHL; 3ZUQ; 4KBB; 5VID; 5VMR; 6G5F; 6G5G; 6G5K; 6QNS; 6UC6; 6UFT; 6UHT; 6UL4; |
| Mapped Pubmed ID | 11679763; 12146945; 14979717; 15781237; 16545378; 21078393; 22139146; 22265973; 28953867; 30746458; 32182233; |
| Motif | MOTIF 1260..1263; /note="Host ganglioside-binding motif"; /evidence="ECO:0000269|PubMed:10932256, ECO:0000305|PubMed:14731268, ECO:0000305|PubMed:17185412" |
| Gene Encoded By | |
| Mass | 150,803 |
| Kinetics | BIOPHYSICOCHEMICAL PROPERTIES: Kinetic parameters: KM=19.2 uM for over-expressed human VAMP1 {ECO:0000269|PubMed:22289120}; KM=29.9 uM for over-expressed human VAMP2 {ECO:0000269|PubMed:22289120}; KM=11.6 uM for over-expressed human VAMP3 {ECO:0000269|PubMed:22289120}; Note=kcat is 3.96, 4.68 and 3.50 sec(-1) for over-expressed human VAMP1, VAMP2 and VAMP3 respectively. {ECO:0000269|PubMed:22289120}; |
| Metal Binding | METAL 230; /note="Zinc; via tele nitrogen; catalytic"; /evidence="ECO:0000255|PROSITE-ProRule:PRU10095, ECO:0000269|PubMed:10932256, ECO:0000305|PubMed:1429690, ECO:0007744|PDB:1EPW, ECO:0007744|PDB:1F31, ECO:0007744|PDB:2NP0"; METAL 234; /note="Zinc; via tele nitrogen; catalytic"; /evidence="ECO:0000255|PROSITE-ProRule:PRU10095, ECO:0000269|PubMed:10932256, ECO:0000305|PubMed:1429690, ECO:0007744|PDB:1EPW, ECO:0007744|PDB:1F31, ECO:0007744|PDB:2NP0"; METAL 268; /note="Zinc; catalytic"; /evidence="ECO:0000269|PubMed:10932256, ECO:0007744|PDB:1EPW, ECO:0007744|PDB:1F31, ECO:0007744|PDB:2NP0" |
| Rhea ID | |
| Cross Reference Brenda | 3.4.24.69; |