| IED ID | IndEnz0002012010 |
| Enzyme Type ID | protease012010 |
| Protein Name |
Toxin B EC 3.4.22.- Cleaved into: Glucosyltransferase TcdB EC 2.4.1.- |
| Gene Name | tcdB toxB |
| Organism | Clostridioides difficile (Peptoclostridium difficile) |
| Taxonomic Lineage | cellular organisms Bacteria Terrabacteria group Firmicutes Clostridia Eubacteriales Peptostreptococcaceae Clostridioides Clostridioides difficile (Peptoclostridium difficile) |
| Enzyme Sequence | MSLVNRKQLEKMANVRFRTQEDEYVAILDALEEYHNMSENTVVEKYLKLKDINSLTDIYIDTYKKSGRNKALKKFKEYLVTEVLELKNNNLTPVEKNLHFVWIGGQINDTAINYINQWKDVNSDYNVNVFYDSNAFLINTLKKTVVESAINDTLESFRENLNDPRFDYNKFFRKRMEIIYDKQKNFINYYKAQREENPELIIDDIVKTYLSNEYSKEIDELNTYIEESLNKITQNSGNDVRNFEEFKNGESFNLYEQELVERWNLAAASDILRISALKEIGGMYLDVDMLPGIQPDLFESIEKPSSVTVDFWEMTKLEAIMKYKEYIPEYTSEHFDMLDEEVQSSFESVLASKSDKSEIFSSLGDMEASPLEVKIAFNSKGIINQGLISVKDSYCSNLIVKQIENRYKILNNSLNPAISEDNDFNTTTNTFIDSIMAEANADNGRFMMELGKYLRVGFFPDVKTTINLSGPEAYAAAYQDLLMFKEGSMNIHLIEADLRNFEISKTNISQSTEQEMASLWSFDDARAKAQFEEYKRNYFEGSLGEDDNLDFSQNIVVDKEYLLEKISSLARSSERGYIHYIVQLQGDKISYEAACNLFAKTPYDSVLFQKNIEDSEIAYYYNPGDGEIQEIDKYKIPSIISDRPKIKLTFIGHGKDEFNTDIFAGFDVDSLSTEIEAAIDLAKEDISPKSIEINLLGCNMFSYSINVEETYPGKLLLKVKDKISELMPSISQDSIIVSANQYEVRINSEGRRELLDHSGEWINKEESIIKDISSKEYISFNPKENKITVKSKNLPELSTLLQEIRNNSNSSDIELEEKVMLTECEINVISNIDTQIVEERIEEAKNLTSDSINYIKDEFKLIESISDALCDLKQQNELEDSHFISFEDISETDEGFSIRFINKETGESIFVETEKTIFSEYANHITEEISKIKGTIFDTVNGKLVKKVNLDTTHEVNTLNAAFFIQSLIEYNSSKESLSNLSVAMKVQVYAQLFSTGLNTITDAAKVVELVSTALDETIDLLPTLSEGLPIIATIIDGVSLGAAIKELSETSDPLLRQEIEAKIGIMAVNLTTATTAIITSSLGIASGFSILLVPLAGISAGIPSLVNNELVLRDKATKVVDYFKHVSLVETEGVFTLLDDKIMMPQDDLVISEIDFNNNSIVLGKCEIWRMEGGSGHTVTDDIDHFFSAPSITYREPHLSIYDVLEVQKEELDLSKDLMVLPNAPNRVFAWETGWTPGLRSLENDGTKLLDRIRDNYEGEFYWRYFAFIADALITTLKPRYEDTNIRINLDSNTRSFIVPIITTEYIREKLSYSFYGSGGTYALSLSQYNMGINIELSESDVWIIDVDNVVRDVTIESDKIKKGDLIEGILSTLSIEENKIILNSHEINFSGEVNGSNGFVSLTFSILEGINAIIEVDLLSKSYKLLISGELKILMLNSNHIQQKIDYIGFNSELQKNIPYSFVDSEGKENGFINGSTKEGLFVSELPDVVLISKVYMDDSKPSFGYYSNNLKDVKVITKDNVNILTGYYLKDDIKISLSLTLQDEKTIKLNSVHLDESGVAEILKFMNRKGNTNTSDSLMSFLESMNIKSIFVNFLQSNIKFILDANFIISGTTSIGQFEFICDENDNIQPYFIKFNTLETNYTLYVGNRQNMIVEPNYDLDDSGDISSTVINFSQKYLYGIDSCVNKVVISPNIYTDEINITPVYETNNTYPEVIVLDANYINEKINVNINDLSIRYVWSNDGNDFILMSTSEENKVSQVKIRFVNVFKDKTLANKLSFNFSDKQDVPVSEIILSFTPSYYEDGLIGYDLGLVSLYNEKFYINNFGMMVSGLIYINDSLYYFKPPVNNLITGFVTVGDDKYYFNPINGGAASIGETIIDDKNYYFNQSGVLQTGVFSTEDGFKYFAPANTLDENLEGEAIDFTGKLIIDENIYYFDDNYRGAVEWKELDGEMHYFSPETGKAFKGLNQIGDYKYYFNSDGVMQKGFVSINDNKHYFDDSGVMKVGYTEIDGKHFYFAENGEMQIGVFNTEDGFKYFAHHNEDLGNEEGEEISYSGILNFNNKIYYFDDSFTAVVGWKDLEDGSKYYFDEDTAEAYIGLSLINDGQYYFNDDGIMQVGFVTINDKVFYFSDSGIIESGVQNIDDNYFYIDDNGIVQIGVFDTSDGYKYFAPANTVNDNIYGQAVEYSGLVRVGEDVYYFGETYTIETGWIYDMENESDKYYFNPETKKACKGINLIDDIKYYFDEKGIMRTGLISFENNNYYFNENGEMQFGYINIEDKMFYFGEDGVMQIGVFNTPDGFKYFAHQNTLDENFEGESINYTGWLDLDEKRYYFTDEYIAATGSVIIDGEEYYFDPDTAQLVISE |
| Enzyme Length | 2366 |
| Uniprot Accession Number | P18177 |
| Absorption | |
| Active Site | ACT_SITE 653; /note="For protease activity"; /evidence="ECO:0000255|PROSITE-ProRule:PRU01107, ECO:0000269|PubMed:27571750"; ACT_SITE 698; /note="Nucleophile; for protease activity"; /evidence="ECO:0000255|PROSITE-ProRule:PRU01107, ECO:0000269|PubMed:27571750" |
| Activity Regulation | ACTIVITY REGULATION: [Toxin B]: Protease activity is activated upon binding to 1D-myo-inositol hexakisphosphate (InsP6), which induces conformational reorganization (PubMed:17334356). Inhibited by bezlotoxumab, also named Zinplava, a monoclonal antibody approved by the Food and Drug Administration (FDA), which specifically targets TcdB (PubMed:24821719). {ECO:0000269|PubMed:17334356, ECO:0000269|PubMed:24821719}. |
| Binding Site | BINDING 139; /note="UDP-alpha-D-glucose"; /evidence="ECO:0000269|PubMed:16054646, ECO:0000269|PubMed:28433497, ECO:0007744|PDB:2BVL, ECO:0007744|PDB:2BVM, ECO:0007744|PDB:5UQM, ECO:0007744|PDB:5UQN"; BINDING 577; /note="1D-myo-inositol hexakisphosphate"; /evidence="ECO:0000250|UniProtKB:P16154"; BINDING 600; /note="1D-myo-inositol hexakisphosphate"; /evidence="ECO:0000250|UniProtKB:P16154"; BINDING 647; /note="1D-myo-inositol hexakisphosphate"; /evidence="ECO:0000250|UniProtKB:P16154"; BINDING 764; /note="1D-myo-inositol hexakisphosphate"; /evidence="ECO:0000250|UniProtKB:P16154"; BINDING 775; /note="1D-myo-inositol hexakisphosphate"; /evidence="ECO:0000250|UniProtKB:P16154"; BINDING 792; /note="1D-myo-inositol hexakisphosphate"; /evidence="ECO:0000250|UniProtKB:P16154" |
| Calcium Binding | |
| catalytic Activity | CATALYTIC ACTIVITY: [Glucosyltransferase TcdB]: Reaction=L-threonyl-[protein] + UDP-alpha-D-glucose = 3-O-(alpha-D-glucosyl)-L-threonyl-[protein] + H(+) + UDP; Xref=Rhea:RHEA:64684, Rhea:RHEA-COMP:11060, Rhea:RHEA-COMP:16656, ChEBI:CHEBI:15378, ChEBI:CHEBI:30013, ChEBI:CHEBI:58223, ChEBI:CHEBI:58885, ChEBI:CHEBI:156085; Evidence={ECO:0000269|PubMed:16157585, ECO:0000269|PubMed:17901056, ECO:0000269|PubMed:24905543, ECO:0000269|PubMed:24919149, ECO:0000269|PubMed:7777059};PhysiologicalDirection=left-to-right; Xref=Rhea:RHEA:64685; Evidence={ECO:0000269|PubMed:16157585, ECO:0000269|PubMed:17901056, ECO:0000269|PubMed:24905543, ECO:0000269|PubMed:24919149, ECO:0000269|PubMed:7777059}; |
| DNA Binding | |
| EC Number | 3.4.22.-; 2.4.1.- |
| Enzyme Function | FUNCTION: [Toxin B]: Precursor of a cytotoxin that targets and disrupts the colonic epithelium, inducing the host inflammatory and innate immune responses and resulting in diarrhea and pseudomembranous colitis (PubMed:20844489, PubMed:24919149). TcdB constitutes the main toxin that mediates the pathology of C.difficile infection, an opportunistic pathogen that colonizes the colon when the normal gut microbiome is disrupted (PubMed:19252482, PubMed:20844489). Compared to TcdA, TcdB is more virulent and more important for inducing the host inflammatory and innate immune responses (PubMed:19252482, PubMed:24919149). This form constitutes the precursor of the toxin: it enters into host cells and mediates autoprocessing to release the active toxin (Glucosyltransferase TcdB) into the host cytosol (PubMed:10768933, PubMed:11152463, PubMed:12941936, PubMed:17334356, PubMed:20498856). Targets colonic epithelia by binding to the frizzled receptors FZD1, FZD2 and FZD7, and enters host cells via clathrin-mediated endocytosis (PubMed:27680706). Frizzled receptors constitute the major host receptors in the colonic epithelium, but other receptors, such as CSPG4 or NECTIN3/PVRL3, have been identified (PubMed:25547119, PubMed:26038560, PubMed:27680706). Binding to carbohydrates and sulfated glycosaminoglycans on host cell surface also contribute to entry into cells (By similarity). Once entered into host cells, acidification in the endosome promotes the membrane insertion of the translocation region and formation of a pore, leading to translocation of the GT44 and peptidase C80 domains across the endosomal membrane (PubMed:11152463, PubMed:12941936, PubMed:24567384). This activates the peptidase C80 domain and autocatalytic processing, releasing the N-terminal part (Glucosyltransferase TcdB), which constitutes the active part of the toxin, in the cytosol (PubMed:17334356, PubMed:27571750). {ECO:0000250|UniProtKB:P16154, ECO:0000269|PubMed:10768933, ECO:0000269|PubMed:11152463, ECO:0000269|PubMed:12941936, ECO:0000269|PubMed:17334356, ECO:0000269|PubMed:19252482, ECO:0000269|PubMed:20498856, ECO:0000269|PubMed:20844489, ECO:0000269|PubMed:24567384, ECO:0000269|PubMed:24919149, ECO:0000269|PubMed:25547119, ECO:0000269|PubMed:26038560, ECO:0000269|PubMed:27571750, ECO:0000269|PubMed:27680706}.; FUNCTION: [Glucosyltransferase TcdB]: Active form of the toxin, which is released into the host cytosol following autoprocessing and inactivates small GTPases (PubMed:8144660, PubMed:7777059, PubMed:16157585, PubMed:17901056, PubMed:24905543, PubMed:24919149). Acts by mediating monoglucosylation of small GTPases of the Rho family (Rac1, RhoA, RhoB, RhoC, RhoG and Cdc42) in host cells at the conserved threonine residue located in the switch I region ('Thr-37/35'), using UDP-alpha-D-glucose as the sugar donor (PubMed:7777059, PubMed:16157585, PubMed:17901056, PubMed:24905543, PubMed:24919149). Monoglucosylation of host small GTPases completely prevents the recognition of the downstream effector, blocking the GTPases in their inactive form, leading to actin cytoskeleton disruption and cell death, resulting in the loss of colonic epithelial barrier function (PubMed:7777059, PubMed:24919149). {ECO:0000269|PubMed:16157585, ECO:0000269|PubMed:17901056, ECO:0000269|PubMed:24905543, ECO:0000269|PubMed:24919149, ECO:0000269|PubMed:7777059, ECO:0000269|PubMed:8144660}. |
| Temperature Dependency | |
| PH Dependency | |
| Pathway | |
| nucleotide Binding | |
| Features | Active site (2); Beta strand (101); Binding site (7); Chain (2); Domain (2); Helix (42); Initiator methionine (1); Metal binding (7); Mutagenesis (34); Region (12); Repeat (18); Site (1); Turn (26) |
| Keywords | 3D-structure;Autocatalytic cleavage;Direct protein sequencing;Enterotoxin;Glycosyltransferase;Host cell membrane;Host cytoplasm;Host endosome;Host membrane;Hydrolase;Lipid-binding;Magnesium;Manganese;Membrane;Metal-binding;Protease;Repeat;Secreted;Thiol protease;Toxin;Transferase;Virulence;Zinc |
| Interact With | |
| Induction | |
| Subcellular Location | SUBCELLULAR LOCATION: [Toxin B]: Secreted {ECO:0000269|PubMed:22685398}. Host endosome membrane {ECO:0000269|PubMed:11152463, ECO:0000305|PubMed:12941936}. Note=Secreted from C.difficile cell into the extracellular environment via help of holin-like protein TcdE/UtxA (PubMed:22685398). Binds to the cell surface receptors via the receptor-binding region and enters the cells via clathrin-mediated endocytosis (PubMed:20498856). Acidification in the endosome triggers conformational changes that promote the membrane insertion of the translocation region, allowing formation of a pore, leading to translocation of the GT44 and peptidase C80 domains across the endosomal membrane (PubMed:10768933, PubMed:11152463, PubMed:12941936). 1D-myo-inositol hexakisphosphate-binding (InsP6) activates the peptidase C80 domain and autoprocessing, generating the Glucosyltransferase TcdB form, which is released in the host cytosol (PubMed:17334356). {ECO:0000269|PubMed:10768933, ECO:0000269|PubMed:11152463, ECO:0000269|PubMed:12941936, ECO:0000269|PubMed:17334356, ECO:0000269|PubMed:20498856, ECO:0000269|PubMed:22685398}.; SUBCELLULAR LOCATION: [Glucosyltransferase TcdB]: Host cytoplasm, host cytosol {ECO:0000269|PubMed:12941936, ECO:0000269|PubMed:15632438}. Host cell membrane {ECO:0000250|UniProtKB:Q46342}; Peripheral membrane protein {ECO:0000250|UniProtKB:Q46342}; Cytoplasmic side {ECO:0000250|UniProtKB:Q46342}. Note=Binding to phospholipids, such as phosphatidylserine and phosphatidic acid promotes localization to the inner face of the cell membrane close to its membrane anchored substrates (small GTPases). {ECO:0000250|UniProtKB:Q46342}. |
| Modified Residue | |
| Post Translational Modification | PTM: [Toxin B]: Undergoes autocatalytic cleavage to release the N-terminal part (Glucosyltransferase TcdB), which constitutes the active part of the toxin, in the host cytosol (PubMed:12941936, PubMed:17334356, PubMed:27571750). 1D-myo-inositol hexakisphosphate-binding (InsP6) activates the peptidase C80 domain and promotes autoprocessing (PubMed:17334356). {ECO:0000269|PubMed:12941936, ECO:0000269|PubMed:17334356, ECO:0000269|PubMed:27571750}. |
| Signal Peptide | |
| Structure 3D | Electron microscopy (2); X-ray crystallography (13) |
| Cross Reference PDB | 2BVL; 2BVM; 4NC2; 4NP4; 5UQM; 5UQN; 5UQT; 6AR6; 6C0B; 6OQ5; 6OQ6; 6OQ7; 6OQ8; 7ML7; 7S0Y; |
| Mapped Pubmed ID | 16730714; 20534589; 26602083; 34145250; |
| Motif | |
| Gene Encoded By | |
| Mass | 269,712 |
| Kinetics | BIOPHYSICOCHEMICAL PROPERTIES: [Glucosyltransferase TcdB]: Kinetic parameters: KM=6 uM for UDP-alpha-D-glucose {ECO:0000269|PubMed:16157585}; KM=4.3 uM for UDP-alpha-D-glucose {ECO:0000269|PubMed:17901056}; KM=960 uM for UDP-N-acetyl-alpha-D-glucosamine {ECO:0000269|PubMed:16157585}; Note=kcat is 2120 h(-1) with UDP-alpha-D-glucose as substrate (PubMed:16157585). kcat is 11 h(-1) with UDP-N-acetyl-alpha-D-glucosamine as substrate (PubMed:16157585). {ECO:0000269|PubMed:16157585}; |
| Metal Binding | METAL 286; /note="Magnesium or manganese"; /evidence="ECO:0000305|PubMed:31308519, ECO:0007744|PDB:6OQ5, ECO:0007744|PDB:6OQ7"; METAL 288; /note="Magnesium or manganese"; /evidence="ECO:0000269|PubMed:16054646, ECO:0000269|PubMed:28433497, ECO:0000305|PubMed:31308519, ECO:0007744|PDB:2BVL, ECO:0007744|PDB:5UQM, ECO:0007744|PDB:5UQN, ECO:0007744|PDB:5UQT, ECO:0007744|PDB:6OQ5, ECO:0007744|PDB:6OQ7"; METAL 515; /note="Magnesium or manganese"; /evidence="ECO:0000269|PubMed:16054646, ECO:0000269|PubMed:28433497, ECO:0000305|PubMed:31308519, ECO:0007744|PDB:2BVL, ECO:0007744|PDB:5UQM, ECO:0007744|PDB:5UQN, ECO:0007744|PDB:5UQT, ECO:0007744|PDB:6OQ5, ECO:0007744|PDB:6OQ7"; METAL 545; /note="Zinc; via carbonyl oxygen"; /evidence="ECO:0000250|UniProtKB:P16154"; METAL 546; /note="Zinc"; /evidence="ECO:0000269|PubMed:31308519, ECO:0007744|PDB:6OQ5"; METAL 653; /note="Zinc"; /evidence="ECO:0000269|PubMed:31308519, ECO:0007744|PDB:6OQ5"; METAL 757; /note="Zinc"; /evidence="ECO:0000269|PubMed:31308519, ECO:0000305|PubMed:27571750, ECO:0007744|PDB:6OQ5" |
| Rhea ID | RHEA:64684; RHEA:64685 |
| Cross Reference Brenda | 2.4.1.B62;3.1.4.B4; |