| IED ID | IndEnz0002007649 |
| Enzyme Type ID | protease007649 |
| Protein Name |
Botulinum neurotoxin type E BoNT/E Bontoxilysin-E Cleaved into: Botulinum neurotoxin E light chain LC EC 3.4.24.69 ; Botulinum neurotoxin E heavy chain HC |
| Gene Name | botE |
| Organism | Clostridium botulinum |
| Taxonomic Lineage | cellular organisms Bacteria Terrabacteria group Firmicutes Clostridia Eubacteriales Clostridiaceae Clostridium Clostridium botulinum |
| Enzyme Sequence | MPKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTSLKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTPDNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHRFGSIAIVTFSPEYSFRFNDNCMNEFIQDPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPLITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYKDVFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLRTKFQVKCRQTYIGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITGRGLVKKIIRFCKNIVSVKGIRKSICIEINNGELFFVASENSYNDDNINTPKEIDDTVTSNNNYENDLDQVILNFNSESAPGLSDEKLNLTIQNDAYIPKYDSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSSIDTALLEQPKIYTFFSSEFINNVNKPVQAALFVSWIQQVLVDFTTEANQKSTVDKIADISIVVPYIGLALNIGNEAQKGNFKDALELLGAGILLEFEPELLIPTILVFTIKSFLGSSDNKNKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKRKEQMYQALQNQVNAIKTIIESKYNSYTLEEKNELTNKYDIKQIENELNQKVSIAMNNIDRFLTESSISYLMKIINEVKINKLREYDENVKTYLLNYIIQHGSILGESQQELNSMVTDTLNNSIPFKLSSYTDDKILISYFNKFFKRIKSSSVLNMRYKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEVNISQNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWKVSLNHNEIIWTFEDNRGINQKLAFNYGNANGISDYINKWIFVTITNDRLGDSKLYINGNLIDQKSILNLGNIHVSDNILFKIVNCSYTRYIGIRYFNIFDKELDETEIQTLYSNEPNTNILKDFWGNYLLYDKEYYLLNVLKPNNFIDRRKDSTLSINNIRSTILLANRLYSGIKVKIQRVNNSSTNDNLVRKNDQVYINFVASKTHLFPLYADTATTNKEKTIKISSSGNRFNQVVVMNSVGNCTMNFKNNNGNNIGLLGFKADTVVASTWYYTHMRDHTNSNGCFWNFISEEHGWQEK |
| Enzyme Length | 1251 |
| Uniprot Accession Number | Q00496 |
| Absorption | |
| Active Site | ACT_SITE 213; /note="Proton acceptor"; /evidence="ECO:0000255|PROSITE-ProRule:PRU10095, ECO:0000305|PubMed:15157097" |
| Activity Regulation | ACTIVITY REGULATION: Proteolysis of SNAP25 by whole toxin inhibited by dipicolinic acid, 1,10-phenanthroline and EDTA (PubMed:8294407). {ECO:0000269|PubMed:8294407}. |
| Binding Site | |
| 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:8243676, ECO:0000269|PubMed:8294407, ECO:0000269|PubMed:9886085}; |
| DNA Binding | |
| EC Number | 3.4.24.69 |
| Enzyme Function | FUNCTION: [Botulinum neurotoxin type E]: Botulinum toxin causes flaccid paralysis by inhibiting neurotransmitter (acetylcholine) release from the presynaptic membranes of nerve terminals of eukaryotic host skeletal and autonomic nervous system, with frequent heart or respiratory failure. Precursor of botulinum neurotoxin E which has 2 coreceptors; complex polysialylated gangliosides found on neural tissue and specific membrane-anchored proteins found in synaptic vesicles. Receptor proteins are exposed on host presynaptic cell membrane during neurotransmitter release, when the toxin heavy chain (HC) binds to them (PubMed:19476346, PubMed:19650874). 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 HC forms pores that allows the light chain (LC) to translocate into the cytosol (PubMed:22720883). 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). Electrical stimulation increases uptake of toxin, probably by transiently exposing a receptor found in eukaryotic target synaptic vesicles (PubMed:19476346, PubMed:19650874). Uses the large lumenal domain of synaptic vesicle glycoproteins 2A and 2B (SV2A and SV2B) but not SV2C as receptor; an N-linked glycan of SV2 is essential for receptor function (PubMed:18815274, PubMed:19476346). Host cell gangliosides are also required for neurotoxin uptake and full toxicity (PubMed:18815274, PubMed:19650874). BoNT/E is a 'coincidence detector'; it requires simultaneous binding to coreceptor GT1b and low pH to transform into a membrane-bound, oligomeric channel (PubMed:22720883). Requires trypsinization and reduction before it can be used in assays in vitro (PubMed:8294407). {ECO:0000250|UniProtKB:P0DPI0, ECO:0000269|PubMed:18815274, ECO:0000269|PubMed:19476346, ECO:0000269|PubMed:19650874, ECO:0000269|PubMed:22720883, ECO:0000305|PubMed:8294407}.; FUNCTION: [Botulinum neurotoxin E light chain]: Has proteolytic activity (PubMed:8243676, PubMed:8294407, PubMed:9886085). After translocation into the eukaryotic host cytosol, inhibits neurotransmitter release by acting as a zinc endopeptidase that catalyzes the hydrolysis of the '180-Arg-|-Ile-181' bond in SNAP25 (PubMed:8243676, PubMed:8294407, PubMed:9886085). Hydrolyzes the '185-Arg-|-Ile-186' bond of mouse SNAP23, but not in human which has a different sequence (PubMed:9886085). Recognizes the '146-Met--Asp-186' region of SNAP25 (PubMed:9886085). The reaction mechanism probably has a nucleophilic water held in place by Glu-213 (PubMed:15157097, PubMed:15938619). Reduction of the interchain disulfide bond occurs in the host cytosol and probably prevents retrotranslocation into the synaptic vesicle (PubMed:17666397). {ECO:0000269|PubMed:15157097, ECO:0000269|PubMed:15938619, ECO:0000269|PubMed:17666397, ECO:0000269|PubMed:8243676, ECO:0000269|PubMed:8294407, ECO:0000269|PubMed:9886085}.; FUNCTION: [Botulinum neurotoxin E heavy chain]: Responsible for host epithelial cell transcytosis, host nerve cell targeting and translocation of light chain (LC) into host cytosol (PubMed:17666397). 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 cell surface (PubMed:10413679). It probably simultaneously recognizes 2 coreceptors; polysialated gangliosides and either of the receptor proteins SV2A and SV2B in close proximity on host synaptic vesicles (PubMed:18815274, PubMed:19650874, PubMed:19476346). The N-terminus of the TD wraps an extended belt around the perimeter of the light chain (LC), protecting Zn(2+) in the active site (PubMed:19118561). The belt 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). Responsible for adherence of the toxin to the cell surface; HC alone prevents uptake of whole toxin by neural cells, and delays paralysis onset by 154% (PubMed:10413679). Significantly decreases uptake and toxicity of whole BoNT/E, but also interferes with uptake of BoNT/C; binds GT1b in vitro (PubMed:19650874). Binds to synaptic vesicle glycoproteins SV2A and SV2B which serve as coreceptors with gangliosides (PubMed:18815274, PubMed:19650874). Interaction with SV2 proteins requires SV2 glycosylation (PubMed:19476346). HC alone significantly decreases uptake and toxicity of whole BoNT/E (PubMed:19650874). HC is responsible for translocation of LC into the host cytosol; an intact disulfide bond between the 2 subunits is required for translocation, which is reduced upon contact with the host cytosol (PubMed:17666397). {ECO:0000250|UniProtKB:P0DPI0, ECO:0000269|PubMed:10413679, ECO:0000269|PubMed:17666397, ECO:0000269|PubMed:18815274, ECO:0000269|PubMed:19476346, ECO:0000269|PubMed:19650874, ECO:0000305|PubMed:17907800}. |
| Temperature Dependency | |
| PH Dependency | |
| Pathway | |
| nucleotide Binding | |
| Features | Active site (1); Beta strand (61); Chain (3); Disulfide bond (1); Helix (38); Initiator methionine (1); Metal binding (3); Motif (1); Mutagenesis (9); Region (5); Sequence conflict (11); Turn (15) |
| 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;Protease;Secreted;Toxin;Transmembrane;Virulence;Zinc |
| Interact With | |
| Induction | |
| Subcellular Location | SUBCELLULAR LOCATION: [Botulinum neurotoxin type E]: Secreted {ECO:0000305|PubMed:8294407}. Note=At pH 4.4 in the presence of ganglioside GT1b, becomes a membrane-associated hydrophobic protein (PubMed:22720883). {ECO:0000269|PubMed:22720883}.; SUBCELLULAR LOCATION: [Botulinum neurotoxin E light chain]: Secreted. Host cytoplasm, host cytosol {ECO:0000305|PubMed:8243676, ECO:0000305|PubMed:8294407}.; SUBCELLULAR LOCATION: [Botulinum neurotoxin E heavy chain]: Secreted. Host cell junction, host synapse, host presynaptic cell membrane {ECO:0000305|PubMed:10413679}. Host cytoplasmic vesicle, host secretory vesicle, host synaptic vesicle membrane {ECO:0000250|UniProtKB:P0DPI0}; Multi-pass membrane protein {ECO:0000305}. |
| Modified Residue | |
| Post Translational Modification | |
| Signal Peptide | |
| Structure 3D | X-ray crystallography (10) |
| Cross Reference PDB | 1T3A; 1T3C; 1ZKW; 1ZKX; 1ZL5; 1ZL6; 1ZN3; 3FFZ; 7K7Y; 7K84; |
| Mapped Pubmed ID | 24583011; 32992561; |
| Motif | MOTIF 1221..1224; /note="Host ganglioside-binding motif"; /evidence="ECO:0000250|UniProtKB:P0DPI0, ECO:0000305|PubMed:19650874" |
| Gene Encoded By | |
| Mass | 143,844 |
| Kinetics | BIOPHYSICOCHEMICAL PROPERTIES: Kinetic parameters: KM=7.9 uM for purified SNAP25 with botulinum neurotoxin E light chain {ECO:0000269|PubMed:15938619}; Note=kcat is 257 min (-1) with botulinum neurotoxin E light chain. {ECO:0000269|PubMed:15938619}; |
| Metal Binding | METAL 212; /note="Zinc; via tele nitrogen; catalytic"; /evidence="ECO:0000255|PROSITE-ProRule:PRU10095, ECO:0007744|PDB:1T3A, ECO:0007744|PDB:1T3C, ECO:0007744|PDB:1ZKW, ECO:0007744|PDB:1ZKX, ECO:0007744|PDB:1ZL6, ECO:0007744|PDB:1ZN3, ECO:0007744|PDB:3FFZ"; METAL 216; /note="Zinc; via tele nitrogen; catalytic"; /evidence="ECO:0000255|PROSITE-ProRule:PRU10095, ECO:0007744|PDB:1T3A, ECO:0007744|PDB:1T3C, ECO:0007744|PDB:1ZKW, ECO:0007744|PDB:1ZKX, ECO:0007744|PDB:1ZL6, ECO:0007744|PDB:1ZN3, ECO:0007744|PDB:3FFZ"; METAL 251; /note="Zinc; catalytic"; /evidence="ECO:0007744|PDB:1T3A, ECO:0007744|PDB:1T3C, ECO:0007744|PDB:1ZKW, ECO:0007744|PDB:1ZKX, ECO:0007744|PDB:1ZL6, ECO:0007744|PDB:1ZN3, ECO:0007744|PDB:3FFZ" |
| Rhea ID | |
| Cross Reference Brenda | 3.4.24.69; |