| IED ID | IndEnz0002005584 |
| Enzyme Type ID | protease005584 |
| Protein Name |
Botulinum neurotoxin type D BoNT/D Bontoxilysin-D Cleaved into: Botulinum neurotoxin D light chain LC EC 3.4.24.69 ; Botulinum neurotoxin D heavy chain HC |
| Gene Name | botD |
| Organism | Clostridium botulinum D phage (Clostridium botulinum D bacteriophage) |
| Taxonomic Lineage | Viruses Duplodnaviria Heunggongvirae Uroviricota Caudoviricetes Caudovirales Siphoviridae (phages with long non-contractile tails) unclassified Siphoviridae Clostridium botulinum D phage (Clostridium botulinum D bacteriophage) |
| Enzyme Sequence | MTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSKPPRPTSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSIFCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFVVNIDKFNSLYSDLTNVMSEVVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIENSGQNIERNPALQKLSSESVVDLFTKVCLRLTKNSRDDSTCIKVKNNRLPYVADKDSISQEIFENKIITDETNVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSYYYLESQKLSNNVENITLTTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKKDTLDKISDVSVIIPYIGPALNIGNSALRGNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREKIIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNIILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFNSINDSKILSLQNKKNALVDTSGYNAEVRVGDNVQLNTIYTNDFKLSSSGDKIIVNLNNNILYSAIYENSSVSFWIKISKDLTNSHNEYTIINSIEQNSGWKLCIRNGNIEWILQDVNRKYKSLIFDYSESLSHTGYTNKWFFVTITNNIMGYMKLYINGELKQSQKIEDLDEVKLDKTIVFGIDENIDENQMLWIRDFNIFSKELSNEDINIVYEGQILRNVIKDYWGNPLKFDTEYYIINDNYIDRYIAPESNVLVLVQYPDRSKLYTGNPITIKSVSDKNPYSRILNGDNIILHMLYNSRKYMIIRDTDTIYATQGGECSQNCVYALKLQSNLGNYGIGIFSIKNIVSKNKYCSQIFSSFRENTMLLADIYKPWRFSFKNAYTPVAVTNYETKLLSTSSFWKFISRDPGWVE |
| Enzyme Length | 1276 |
| Uniprot Accession Number | P19321 |
| Absorption | |
| Active Site | ACT_SITE 230; /evidence=ECO:0000255|PROSITE-ProRule:PRU10095 |
| Activity Regulation | ACTIVITY REGULATION: [Botulinum neurotoxin D light chain]: Inhibited by dipicolinic acid, captopril, 1,10-phenanthroline and EDTA. {ECO:0000269|PubMed:8175689}. |
| Binding Site | BINDING 1192; /note="N-acetyl-beta-neuraminic acid"; /evidence="ECO:0000269|PubMed:20704566, ECO:0007744|PDB:3OBT"; BINDING 1239; /note="N-acetyl-beta-neuraminic acid"; /evidence="ECO:0000269|PubMed:20704566, ECO:0007744|PDB:3OBT" |
| 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:8175689, ECO:0000269|PubMed:8197120}; |
| DNA Binding | |
| EC Number | 3.4.24.69 |
| Enzyme Function | FUNCTION: [Botulinum neurotoxin type D]: 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 (PubMed:8175689, PubMed:16252491). Precursor of botulinum neurotoxin D for which a proteinaceous coreceptor is controversial. In double SV2A/SV2B knockout mice this toxin does not degrade its synaptobrevin target; introducing SV2A, SV2B or SV2C restores target cleavage (PubMed:21483489). Recognition of SV2 by this toxin does not occur via SV2 glycosylation or its large extracellular loop 4 (PubMed:21483489). Another group does not find a convincing interaction with SV2 (PubMed:21632541). Thus a protein receptor for this BoNT serotype has yet to be definitively proven. Recognizes at least 1 complex polysialylated ganglioside found on neural tissue. Electrical stimulation increases uptake of toxin in an ex vivo assay, presumably by transiently exposing a receptor usually found in eukaryotic target synaptic vesicles (PubMed:19650874, PubMed:21483489, PubMed:21632541). Upon synaptic vesicle recycling the toxin is taken up via the endocytic pathway; when the pH of the toxin-containing endosome drops a structural rearrangement occurs so that the N-terminus of the heavy chain (HC) forms pores that allows the light chain (LC) to translocate into the cytosol (By similarity). 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 (By similarity). Requires complex eukaryotic host polysialogangliosides for full neurotoxicity and for binding to neurons (PubMed:20704566, PubMed:21483489). {ECO:0000250|UniProtKB:P0DPI0, ECO:0000269|PubMed:16252491, ECO:0000269|PubMed:19650874, ECO:0000269|PubMed:20704566, ECO:0000269|PubMed:21483489, ECO:0000269|PubMed:21632541, ECO:0000269|PubMed:8175689, ECO:0000305}.; FUNCTION: [Botulinum neurotoxin D light chain]: Has proteolytic activity (PubMed:8175689, PubMed:8197120). After translocation into the eukaryotic host cytosol, inhibits neurotransmitter release by acting as a zinc endopeptidase that cleaves the '61-Lys-|-Leu-62' bond of synaptobrevin-1 (VAMP1), and the equivalent 'Lys-|-Leu' sites in VAMP2 and VAMP3 (PubMed:8175689). Cleaves the '49-Lys-|-Ile-50' bond of A.californica synaptobrevin (AC P35589) (PubMed:8197120). This chain probably has to be partially unfolded to translocate into the eukaryotic host cell cytosol (PubMed:15584922). {ECO:0000269|PubMed:8175689, ECO:0000269|PubMed:8197120, ECO:0000305|PubMed:15584922}.; FUNCTION: [Botulinum neurotoxin D heavy chain]: Responsible for host epithelial cell transcytosis, host nerve cell targeting and translocation of light chain (LC) into eukaryotic host cell cytosol. Composed of 3 subdomains; the translocation domain (TD), and N-terminus and C-terminus of the receptor-binding domain (RBD). The RBD is responsible for the adherence of the toxin to the eukaryotic target cell surface. The N-terminus of the TD wraps an extended belt around the perimeter of the LC, protecting Zn(2+) in the active site; it may also prevent premature LC dissociation from the translocation channel and protect toxin prior to translocation (PubMed:17907800). The TD inserts into synaptic vesicle membrane to allow translocation into the host cytosol (By similarity). The RBD binds eukaryotic host phosphatidylethanolamine, which may serve as toxin receptor (PubMed:16115873). Treatment of synaptosomes with proteinase K does not reduce HC binding, suggesting there is no protein receptor or it is protected from extracellular proteases (PubMed:16115873). HC significantly decreases uptake and toxicity of whole BoNT/D (PubMed:19650874, PubMed:21483489). HC also interferes with uptake of tetanus toxin (PubMed:19650874). Has 2 closely located carbohydrate-binding receptor sites and binds at least 1 GT1b ganglioside (PubMed:20704566). Bind gangliosides in the order GD2 > GT1b > GD1b (PubMed:21632541). Interacts with eukaryotic target protein SV2B (synaptic vesicle glycoprotein 2B) (PubMed:21483489). Expression of SV2A, SV2B or SV2C in mice knocked-out for the SV2 proteins restores entry of BoNT/D and cleavage of VAMP2, suggesting SV2 acts as its receptor (PubMed:21483489). Unlike BoNT/A and BoNT/E, toxin uptake is not mediated by large extracellular loop 4 of SV2 (PubMed:21483489). Another group finds very poor interaction with SV2 proteins, suggesting the possible protein receptor may not have been identified (PubMed:21632541). {ECO:0000250|UniProtKB:P0DPI0, ECO:0000269|PubMed:16115873, ECO:0000269|PubMed:19650874, ECO:0000269|PubMed:20704566, ECO:0000269|PubMed:21483489, ECO:0000269|PubMed:21632541, ECO:0000305|PubMed:17907800}. |
| Temperature Dependency | |
| PH Dependency | |
| Pathway | |
| nucleotide Binding | |
| Features | Active site (1); Beta strand (59); Binding site (2); Chain (3); Disulfide bond (1); Helix (40); Initiator methionine (1); Metal binding (3); Motif (1); Mutagenesis (19); Natural variant (9); Region (6); Turn (15) |
| Keywords | 3D-structure;Direct protein sequencing;Disulfide bond;Hydrolase;Lipid-binding;Metal-binding;Metalloprotease;Neurotoxin;Protease;Secreted;Toxin;Virulence;Zinc |
| Interact With | |
| Induction | |
| Subcellular Location | SUBCELLULAR LOCATION: [Botulinum neurotoxin type D]: Secreted {ECO:0000269|PubMed:11713244, ECO:0000269|PubMed:17581814, ECO:0000269|PubMed:2668193}.; SUBCELLULAR LOCATION: [Botulinum neurotoxin D light chain]: Secreted {ECO:0000269|PubMed:16252491, ECO:0000269|PubMed:8569530}. Note=In animals that have ingested BoNT/D LC acts in the eukaryotic host cytosol (Probable). {ECO:0000305|PubMed:15584922, ECO:0000305|PubMed:8175689}.; SUBCELLULAR LOCATION: [Botulinum neurotoxin D heavy chain]: Secreted {ECO:0000269|PubMed:16252491, ECO:0000269|PubMed:8569530}. Note=Upon incubation with hippocampal cell colocalizes with SV2C, probably in host synaptic vesicles (PubMed:21483489). Upon incubation with primary neurons HC colocalizes with synaptophysin probably in synaptic vesicles of presynaptic cells; a small portion also colocalizes with RAB5 and may be in synaptic vesicle protein sorting endosomes (PubMed:21632541). Probably integrates into the eukaryotic host synaptic vesicle membrane. {ECO:0000269|PubMed:21483489, ECO:0000269|PubMed:21632541, ECO:0000305}. |
| Modified Residue | |
| Post Translational Modification | |
| Signal Peptide | |
| Structure 3D | X-ray crystallography (9) |
| Cross Reference PDB | 2FPQ; 3N7J; 3OBR; 3OBT; 3OGG; 3RMX; 3RMY; 5BQM; 5BQN; |
| Mapped Pubmed ID | 20858456; |
| Motif | MOTIF 1252..1255; /note="Host ganglioside-binding motif"; /evidence="ECO:0000250|UniProtKB:P0DPI0, ECO:0000305|PubMed:20704566" |
| Gene Encoded By | |
| Mass | 146,872 |
| Kinetics | BIOPHYSICOCHEMICAL PROPERTIES: Kinetic parameters: KM=28.9 uM for over-expressed human VAMP1 {ECO:0000269|PubMed:22289120}; KM=28.0 uM for over-expressed human VAMP2 {ECO:0000269|PubMed:22289120}; KM=11.1 uM for over-expressed human VAMP3 {ECO:0000269|PubMed:22289120}; Note=kcat is 0.59, 146.60 and 113.41 sec(-1) for over-expressed human VAMP1, VAMP2 and VAMP3 respectively. {ECO:0000269|PubMed:22289120}; |
| Metal Binding | METAL 229; /note="Zinc; via tele nitrogen; catalytic"; /evidence="ECO:0000269|PubMed:16519520, ECO:0000269|PubMed:26324071, ECO:0007744|PDB:2FPQ, ECO:0007744|PDB:5BQM"; METAL 233; /note="Zinc; via tele nitrogen; catalytic"; /evidence="ECO:0000269|PubMed:16519520, ECO:0000269|PubMed:26324071, ECO:0007744|PDB:2FPQ, ECO:0007744|PDB:5BQM"; METAL 269; /note="Zinc; catalytic"; /evidence="ECO:0000269|PubMed:16519520, ECO:0000269|PubMed:26324071, ECO:0007744|PDB:2FPQ, ECO:0007744|PDB:5BQM" |
| Rhea ID | |
| Cross Reference Brenda | 3.4.24.69; |