| IED ID | IndEnz0002011071 |
| Enzyme Type ID | protease011071 |
| Protein Name |
Toxin A EC 3.4.22.- Cleaved into: Glucosyltransferase TcdA EC 2.4.1.- |
| Gene Name | tcdA toxA |
| Organism | Clostridioides difficile (Peptoclostridium difficile) |
| Taxonomic Lineage | cellular organisms Bacteria Terrabacteria group Firmicutes Clostridia Eubacteriales Peptostreptococcaceae Clostridioides Clostridioides difficile (Peptoclostridium difficile) |
| Enzyme Sequence | MSLISKEELIKLAYSIRPRENEYKTILTNLDEYNKLTTNNNENKYLQLKKLNESIDVFMNKYKTSSRNRALSNLKKDILKEVILIKNSNTSPVEKNLHFVWIGGEVSDIALEYIKQWADINAEYNIKLWYDSEAFLVNTLKKAIVESSTTEALQLLEEEIQNPQFDNMKFYKKRMEFIYDRQKRFINYYKSQINKPTVPTIDDIIKSHLVSEYNRDETVLESYRTNSLRKINSNHGIDIRANSLFTEQELLNIYSQELLNRGNLAAASDIVRLLALKNFGGVYLDVDMLPGIHSDLFKTISRPSSIGLDRWEMIKLEAIMKYKKYINNYTSENFDKLDQQLKDNFKLIIESKSEKSEIFSKLENLNVSDLEIKIAFALGSVINQALISKQGSYLTNLVIEQVKNRYQFLNQHLNPAIESDNNFTDTTKIFHDSLFNSATAENSMFLTKIAPYLQVGFMPEARSTISLSGPGAYASAYYDFINLQENTIEKTLKASDLIEFKFPENNLSQLTEQEINSLWSFDQASAKYQFEKYVRDYTGGSLSEDNGVDFNKNTALDKNYLLNNKIPSNNVEEAGSKNYVHYIIQLQGDDISYEATCNLFSKNPKNSIIIQRNMNESAKSYFLSDDGESILELNKYRIPERLKNKEKVKVTFIGHGKDEFNTSEFARLSVDSLSNEISSFLDTIKLDISPKNVEVNLLGCNMFSYDFNVEETYPGKLLLSIMDKITSTLPDVNKNSITIGANQYEVRINSEGRKELLAHSGKWINKEEAIMSDLSSKEYIFFDSIDNKLKAKSKNIPGLASISEDIKTLLLDASVSPDTKFILNNLKLNIESSIGDYIYYEKLEPVKNIIHNSIDDLIDEFNLLENVSDELYELKKLNNLDEKYLISFEDISKNNSTYSVRFINKSNGESVYVETEKEIFSKYSEHITKEISTIKNSIITDVNGNLLDNIQLDHTSQVNTLNAAFFIQSLIDYSSNKDVLNDLSTSVKVQLYAQLFSTGLNTIYDSIQLVNLISNAVNDTINVLPTITEGIPIVSTILDGINLGAAIKELLDEHDPLLKKELEAKVGVLAINMSLSIAATVASIVGIGAEVTIFLLPIAGISAGIPSLVNNELILHDKATSVVNYFNHLSESKKYGPLKTEDDKILVPIDDLVISEIDFNNNSIKLGTCNILAMEGGSGHTVTGNIDHFFSSPSISSHIPSLSIYSAIGIETENLDFSKKIMMLPNAPSRVFWWETGAVPGLRSLENDGTRLLDSIRDLYPGKFYWRFYAFFDYAITTLKPVYEDTNIKIKLDKDTRNFIMPTITTNEIRNKLSYSFDGAGGTYSLLLSSYPISTNINLSKDDLWIFNIDNEVREISIENGTIKKGKLIKDVLSKIDINKNKLIIGNQTIDFSGDIDNKDRYIFLTCELDDKISLIIEINLVAKSYSLLLSGDKNYLISNLSNTIEKINTLGLDSKNIAYNYTDESNNKYFGAISKTSQKSIIHYKKDSKNILEFYNDSTLEFNSKDFIAEDINVFMKDDINTITGKYYVDNNTDKSIDFSISLVSKNQVKVNGLYLNESVYSSYLDFVKNSDGHHNTSNFMNLFLDNISFWKLFGFENINFVIDKYFTLVGKTNLGYVEFICDNNKNIDIYFGEWKTSSSKSTIFSGNGRNVVVEPIYNPDTGEDISTSLDFSYEPLYGIDRYINKVLIAPDLYTSLININTNYYSNEYYPEIIVLNPNTFHKKVNINLDSSSFEYKWSTEGSDFILVRYLEESNKKILQKIRIKGILSNTQSFNKMSIDFKDIKKLSLGYIMSNFKSFNSENELDRDHLGFKIIDNKTYYYDEDSKLVKGLININNSLFYFDPIEFNLVTGWQTINGKKYYFDINTGAALTSYKIINGKHFYFNNDGVMQLGVFKGPDGFEYFAPANTQNNNIEGQAIVYQSKFLTLNGKKYYFDNNSKAVTGWRIINNEKYYFNPNNAIAAVGLQVIDNNKYYFNPDTAIISKGWQTVNGSRYYFDTDTAIAFNGYKTIDGKHFYFDSDCVVKIGVFSTSNGFEYFAPANTYNNNIEGQAIVYQSKFLTLNGKKYYFDNNSKAVTGLQTIDSKKYYFNTNTAEAATGWQTIDGKKYYFNTNTAEAATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLFEIDGVIYFFGVDGVKAPGIYG |
| Enzyme Length | 2710 |
| Uniprot Accession Number | P16154 |
| Absorption | |
| Active Site | ACT_SITE 655; /note="For protease activity"; /evidence="ECO:0000255|PROSITE-ProRule:PRU01107, ECO:0000269|PubMed:19553670, ECO:0000269|PubMed:27571750"; ACT_SITE 700; /note="Nucleophile; for protease activity"; /evidence="ECO:0000255|PROSITE-ProRule:PRU01107, ECO:0000269|PubMed:19553670, ECO:0000269|PubMed:27571750" |
| Activity Regulation | ACTIVITY REGULATION: [Toxin A]: Protease activity is activated upon binding to 1D-myo-inositol hexakisphosphate (InsP6), which induces conformational reorganization. {ECO:0000269|PubMed:17334356, ECO:0000269|PubMed:19553670}. |
| Binding Site | BINDING 138; /note="UDP-alpha-D-glucose"; /evidence="ECO:0000269|PubMed:22267739, ECO:0000269|PubMed:28433497, ECO:0007744|PDB:3SRZ, ECO:0007744|PDB:5UQK, ECO:0007744|PDB:5UQL"; BINDING 551; /note="1D-myo-inositol hexakisphosphate"; /evidence="ECO:0000269|PubMed:19553670, ECO:0007744|PDB:3HO6"; BINDING 579; /note="1D-myo-inositol hexakisphosphate"; /evidence="ECO:0000269|PubMed:19553670, ECO:0007744|PDB:3HO6"; BINDING 602; /note="1D-myo-inositol hexakisphosphate"; /evidence="ECO:0000269|PubMed:19553670, ECO:0007744|PDB:3HO6"; BINDING 649; /note="1D-myo-inositol hexakisphosphate"; /evidence="ECO:0000269|PubMed:19553670, ECO:0007744|PDB:3HO6"; BINDING 766; /note="1D-myo-inositol hexakisphosphate"; /evidence="ECO:0000269|PubMed:19553670, ECO:0007744|PDB:3HO6"; BINDING 777; /note="1D-myo-inositol hexakisphosphate"; /evidence="ECO:0000269|PubMed:19553670, ECO:0007744|PDB:3HO6"; BINDING 794; /note="1D-myo-inositol hexakisphosphate"; /evidence="ECO:0000269|PubMed:19553670, ECO:0007744|PDB:3HO6"; BINDING 2540; /note="alpha-D-galactosyl-(1->3)-beta-D-galactosyl-(1->4)-N-acetyl-beta-D-glucosamine 1"; /evidence="ECO:0000269|PubMed:16622409, ECO:0007744|PDB:2G7C"; BINDING 2631; /note="alpha-D-galactosyl-(1->3)-beta-D-galactosyl-(1->4)-N-acetyl-beta-D-glucosamine 2"; /evidence="ECO:0000269|PubMed:16622409, ECO:0007744|PDB:2G7C" |
| Calcium Binding | |
| catalytic Activity | CATALYTIC ACTIVITY: [Glucosyltransferase TcdA]: 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:22267739, ECO:0000269|PubMed:22747490, ECO:0000269|PubMed:24905543, ECO:0000269|PubMed:30622517, ECO:0000269|PubMed:7775453};PhysiologicalDirection=left-to-right; Xref=Rhea:RHEA:64685; Evidence={ECO:0000269|PubMed:22267739, ECO:0000269|PubMed:22747490, ECO:0000269|PubMed:24905543, ECO:0000269|PubMed:30622517, ECO:0000269|PubMed:7775453}; |
| DNA Binding | |
| EC Number | 3.4.22.-; 2.4.1.- |
| Enzyme Function | FUNCTION: [Toxin A]: 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). TcdA and TcdB constitute the main toxins that mediate 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 TcdB, TcdA is less virulent and less important for inducing the host inflammatory and innate immune responses (PubMed:19252482). This form constitutes the precursor of the toxin: it enters into host cells and mediates autoprocessing to release the active toxin (Glucosyltransferase TcdA) into the host cytosol (By similarity). Targets colonic epithelia by binding to some receptor, and enters host cells via clathrin-mediated endocytosis (By similarity). Binding to LDLR, as well as carbohydrates and sulfated glycosaminoglycans on host cell surface contribute to entry into cells (PubMed:1670930, PubMed:31160825, PubMed:16622409). In contrast to TcdB, Frizzled receptors FZD1, FZD2 and FZD7 do not act as host receptors in the colonic epithelium for TcdA (PubMed:27680706). 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 (By similarity). This activates the peptidase C80 domain and autocatalytic processing, releasing the N-terminal part (Glucosyltransferase TcdA), which constitutes the active part of the toxin, in the cytosol (PubMed:17334356, PubMed:19553670, PubMed:27571750). {ECO:0000250|UniProtKB:P18177, ECO:0000269|PubMed:16622409, ECO:0000269|PubMed:1670930, ECO:0000269|PubMed:17334356, ECO:0000269|PubMed:19252482, ECO:0000269|PubMed:19553670, ECO:0000269|PubMed:20844489, ECO:0000269|PubMed:27571750, ECO:0000269|PubMed:27680706, ECO:0000269|PubMed:31160825}.; FUNCTION: [Glucosyltransferase TcdA]: Active form of the toxin, which is released into the host cytosol following autoprocessing and inactivates small GTPases (PubMed:7775453, PubMed:24905543, PubMed:30622517, PubMed:22747490, PubMed:22267739). Acts by mediating monoglucosylation of small GTPases of the Rho family (Rac1, RhoA, RhoB, RhoC, Rap2A 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:7775453, PubMed:24905543, PubMed:30622517, PubMed:22747490, PubMed:22267739). 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:7775453). Also able to catalyze monoglucosylation of some members of the Ras family (H-Ras/HRAS, K-Ras/KRAS and N-Ras/NRAS), but with much less efficiency than with Rho proteins, suggesting that it does not act on Ras proteins in vivo (PubMed:30622517). {ECO:0000269|PubMed:22267739, ECO:0000269|PubMed:22747490, ECO:0000269|PubMed:24905543, ECO:0000269|PubMed:30622517, ECO:0000269|PubMed:7775453}. |
| Temperature Dependency | |
| PH Dependency | |
| Pathway | |
| nucleotide Binding | |
| Features | Active site (2); Beta strand (121); Binding site (10); Chain (2); Domain (2); Helix (59); Metal binding (7); Mutagenesis (6); Region (14); Repeat (32); Sequence conflict (1); Site (1); Turn (22) |
| Keywords | 3D-structure;Autocatalytic cleavage;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 A]: Secreted {ECO:0000269|PubMed:22685398}. Host endosome membrane {ECO:0000250|UniProtKB:P18177}. 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 (By similarity). 1D-myo-inositol hexakisphosphate-binding (InsP6) activates the peptidase C80 domain and autoprocessing, generating the Glucosyltransferase TcdA form, which is released in the host cytosol (PubMed:19553670). {ECO:0000250|UniProtKB:P18177, ECO:0000269|PubMed:19553670, ECO:0000269|PubMed:20498856, ECO:0000269|PubMed:22685398}.; SUBCELLULAR LOCATION: [Glucosyltransferase TcdA]: Host cytoplasm, host cytosol {ECO:0000250|UniProtKB:P18177}. 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 A]: Undergoes autocatalytic cleavage to release the N-terminal part (Glucosyltransferase TcdA), which constitutes the active part of the toxin, in the host cytosol (PubMed:17334356, PubMed:22267739, PubMed:19553670, PubMed:27571750). 1D-myo-inositol hexakisphosphate-binding (InsP6) activates the peptidase C80 domain and promotes autoprocessing (PubMed:17334356, PubMed:19553670). {ECO:0000269|PubMed:17334356, ECO:0000269|PubMed:19553670, ECO:0000269|PubMed:22267739, ECO:0000269|PubMed:27571750}. |
| Signal Peptide | |
| Structure 3D | X-ray crystallography (14) |
| Cross Reference PDB | 2F6E; 2G7C; 2QJ6; 3HO6; 3SRZ; 3SS1; 4DMV; 4DMW; 4NBX; 4NBZ; 4R04; 5UMI; 5UQK; 5UQL; |
| Mapped Pubmed ID | - |
| Motif | |
| Gene Encoded By | |
| Mass | 308,056 |
| Kinetics | BIOPHYSICOCHEMICAL PROPERTIES: [Glucosyltransferase TcdA]: Kinetic parameters: KM=43.9 uM for UDP-alpha-D-glucose (in presence of K(+)) {ECO:0000269|PubMed:22747490}; KM=36.3 uM for UDP-alpha-D-glucose (in presence of NH4(+)) {ECO:0000269|PubMed:22747490}; KM=51.1 uM for UDP-alpha-D-glucose (in presence of Na(+)) {ECO:0000269|PubMed:22747490}; Vmax=252.2 pmol/min/ug enzyme with UDP-alpha-D-glucose (in presence of K(+)) {ECO:0000269|PubMed:22747490}; Vmax=162.8 pmol/min/ug enzyme with UDP-alpha-D-glucose (in presence of NH4(+)) {ECO:0000269|PubMed:22747490}; Vmax=36.4 pmol/min/ug enzyme with UDP-alpha-D-glucose (in presence of Na(+)) {ECO:0000269|PubMed:22747490}; |
| Metal Binding | METAL 285; /note="Manganese"; /evidence="ECO:0000269|PubMed:22267739, ECO:0007744|PDB:3SS1"; METAL 287; /note="Manganese"; /evidence="ECO:0000269|PubMed:22267739, ECO:0000269|PubMed:22747490, ECO:0000269|PubMed:28433497, ECO:0007744|PDB:3SRZ, ECO:0007744|PDB:3SS1, ECO:0007744|PDB:5UQK, ECO:0007744|PDB:5UQL"; METAL 514; /note="Manganese"; /evidence="ECO:0000269|PubMed:22267739, ECO:0000269|PubMed:22747490, ECO:0000269|PubMed:28433497, ECO:0007744|PDB:3SRZ, ECO:0007744|PDB:3SS1, ECO:0007744|PDB:5UQK, ECO:0007744|PDB:5UQL"; METAL 544; /note="Zinc; via carbonyl oxygen"; /evidence="ECO:0000269|PubMed:27571750, ECO:0007744|PDB:4R04"; METAL 545; /note="Zinc"; /evidence="ECO:0000269|PubMed:27571750, ECO:0007744|PDB:4R04"; METAL 655; /note="Zinc"; /evidence="ECO:0000269|PubMed:27571750, ECO:0007744|PDB:4R04"; METAL 759; /note="Zinc"; /evidence="ECO:0000269|PubMed:27571750, ECO:0007744|PDB:4R04" |
| Rhea ID | RHEA:64684; RHEA:64685 |
| Cross Reference Brenda | 2.4.1.B62;3.1.4.B4; |