| IED ID | IndEnz0002013675 |
| Enzyme Type ID | protease013675 |
| Protein Name |
Toxin A EC 3.4.22.- Cleaved into: N-acetylglucosaminyltransferase TcdA EC 2.4.1.- |
| Gene Name | tcdA toxA |
| Organism | Clostridium novyi |
| Taxonomic Lineage | cellular organisms Bacteria Terrabacteria group Firmicutes Clostridia Eubacteriales Clostridiaceae Clostridium Clostridium novyi |
| Enzyme Sequence | MLITREQLMKIASIPLKRKEPEYNLILDALENFNRDIEGTSVKEIYSKLSKLNELVDNYQTKYPSSGRNLALENFRDSLYSELRELIKNSRTSTIASKNLSFIWIGGPISDQSLEYYNMWKMFNKDYNIRLFYDKNSLLVNTLKTAIIQESSKVIIEQNQSNILDGTYGHNKFYSDRMKLIYRYKRELKMLYENMKQNNSVDDIIINFLSNYFKYDIGKLNNQKENNNNKMIAIGATDINTENILTNKLKSYYYQELIQTNNLAAASDILRIAILKKYGGVYCDLDFLPGVNLSLFNDISKPNGMDSNYWEAAIFEAIANEKKLMNNYPYKYMEQVPSEIKERILSFVRNHDINDLILPLGDIKISQLEILLSRLKAATGKKTFSNAFIISNNDSLTLNNLISQLENRYEILNSIIQEKFKICETYDSYINSVSELVLETTPKNLSMDGSSFYQQIIGYLSSGFKPEVNSTVFFSGPNIYSSATCDTYHFIKNTFDMLSSQNQEIFEASNNLYFSKTHDEFKSSWLLRSNIAEKEFQKLIKTYIGRTLNYEDGLNFNKWKRVTTSELLKVIEEVNSTKIYENYDLNMILQIQGDDISYESAVNVFGKNPNKSILIQGVDDFANVFYFENGIVQSDNINNILSRFNDIKKIKLTLIGHGENVFNPKLFGGKTVNDLYTNIIKPKLQHLLEREGVILKNKYLKINILGCYMFTPKVDINSTFVGKLFNKISRDLQPKGFSKNQLEISANKYAIRINREGKREVLDYFGKWVSNTDLIAEQISNKYVVYWNEVENTLSARVEQLNKVAEFAKDINSIIQTTNNQELKQSLVNTYADLITTLYSELLKEDIPFELDNIQIKERIILNEISRLHDFSNIILDFYQKNNISNNMIILFDSIIKEKDYYNVKLANKITGETSVIKTYSDSLWNFTNKYKKIVDDIKGIIVKDINGEFIKKADFEIEQNPSLLNSAMLMQLLIDYKPYTEILTNMNTSLKVQAYAQIFQLSIGAIQEATEIVTIISDALNANFNILSKLKVGSSVASVIIDGINLIAALTELKNVKTNFERKLIEAKVGMYSIGFILESSSLISGLLGATAVSEILGVISVPVAGILVGLPSLVNNILVLGEKYNQILDYFSKFYPIVGKNPFSIQDNIIIPYDDIAITELNFKYNKFKYGYAKISGLKVGLVTHIGENIDHYFSAPSLDHYIELSIYPALKLNDTNLPKGNVVLLPSGLNKVYKPEISAIAGANSQEGNGVEVLNLIRNYYVDSNGNTKFPWKYEAPFEYSFSYMRVEYFDTKVNVILDNENKTLIIPVLTIDEMRNKISYEILGDGGQYNVILPVNQTNINIVSNKNDIWNFDVSYIVKESKIEDNKFVLDGFINNIFSTLKVSNDGFKIGKQFISIKNTPRAINLSFKINNNIVIVSIYLNHEKSNSITIISSDLNDIKNNFDNLLDNINYIGLGSISDNTINCIVRNDEVYMEGKIFLNEKKLVFIQNELELHLYDSVNKDSQYLINNPINNVVKYKDGYIVEGTFLINSTENKYSLYIENNKIMLKGLYLESSVFKTIQDKIYSKEKVNDYILSLIKKFFTVNIQLCPFMIVSGVDENNRYLEYMLSTNNKWIINGGYWENDFNNYKIVDFEKCNVIVSGSNKLNSEGDLADTIDVLDKDLENLYIDSVIIIPKVYTKKIIIHPIPNNPQINIINTQSIHDKCHLIIDSVLTNNYHWESDGDDLIITNGLDINIRILQGLSFGFKYKNIYLKFSNYDELSLNDFLLQNYNVKGLYYINGELHYKNIPGDTFEYGWINIDSRWYFFDSINLIAKKGYQEIEGERYYFNPNTGVQESGVFLTPNGLEYFTNKHASSKRWGRAINYTGWLTLDGNKYYFQSNSKAVTGLQKISDKYYYFNDNGQMQIKWQIINNNKYYFDGNTGEAIIGWFNNNKERYYFDSEGRLLTGYQVIGDKSYYFSDNINGNWEEGSGVLKSGIFKTPSGFKLFSSEGDKSAINYKGWLDLNGNKYYFNSDSIAVTGSYNIKGIQYYFNPKTAVLTNGWYTLDNNNYYVSNGHNVLGYQDIDGKGYYFDPSTGIQKAGVFPTPNGLRYFTMKPIDGQRWGQCIDYTGWLHLNGNKYYFGYYNSAVTGWRVLGGKRYFFNIKTGAATTGLLTLSGKRYYFNEKGEQLTLV |
| Enzyme Length | 2178 |
| Uniprot Accession Number | Q46149 |
| Absorption | |
| Active Site | ACT_SITE 657; /note=For protease activity; /evidence=ECO:0000255|PROSITE-ProRule:PRU01107; ACT_SITE 707; /note=Nucleophile; for protease activity; /evidence=ECO:0000255|PROSITE-ProRule:PRU01107 |
| Activity Regulation | ACTIVITY REGULATION: [Toxin A]: Protease activity is activated upon binding to 1D-myo-inositol hexakisphosphate (InsP6), which induces conformational reorganization. {ECO:0000250|UniProtKB:P16154}. |
| Binding Site | BINDING 141; /note=UDP-N-acetyl-alpha-D-glucosamine; /evidence=ECO:0000250|UniProtKB:P18177; BINDING 557; /note=1D-myo-inositol hexakisphosphate; /evidence=ECO:0000250|UniProtKB:P16154; BINDING 607; /note=1D-myo-inositol hexakisphosphate; /evidence=ECO:0000250|UniProtKB:P16154; BINDING 651; /note=1D-myo-inositol hexakisphosphate; /evidence=ECO:0000250|UniProtKB:P16154; BINDING 782; /note=1D-myo-inositol hexakisphosphate; /evidence=ECO:0000250|UniProtKB:P16154 |
| Calcium Binding | |
| catalytic Activity | CATALYTIC ACTIVITY: [N-acetylglucosaminyltransferase TcdA]: Reaction=L-threonyl-[protein] + UDP-N-acetyl-alpha-D-glucosamine = 3-O-(N-acetyl-alpha-D-glucosaminyl)-L-threonyl-[protein] + H(+) + UDP; Xref=Rhea:RHEA:64688, Rhea:RHEA-COMP:11060, Rhea:RHEA-COMP:16657, ChEBI:CHEBI:15378, ChEBI:CHEBI:30013, ChEBI:CHEBI:57705, ChEBI:CHEBI:58223, ChEBI:CHEBI:156086; Evidence={ECO:0000269|PubMed:16157585, ECO:0000269|PubMed:8810274};PhysiologicalDirection=left-to-right; Xref=Rhea:RHEA:64689; Evidence={ECO:0000269|PubMed:16157585, ECO:0000269|PubMed:8810274}; |
| DNA Binding | |
| EC Number | 3.4.22.-; 2.4.1.- |
| Enzyme Function | FUNCTION: [Toxin A]: Precursor of a cytotoxin, which enters into host cells and mediates autoprocessing to release the active toxin (N-acetylglucosaminyltransferase TcdA) into the host cytosol (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 (By similarity). This activates the peptidase C80 domain and autocatalytic processing, releasing the N-terminal part (N-acetylglucosaminyltransferase TcdA), which constitutes the active part of the toxin, in the cytosol (PubMed:17334356). {ECO:0000250|UniProtKB:P18177, ECO:0000269|PubMed:17334356}.; FUNCTION: [N-acetylglucosaminyltransferase TcdA]: Active form of the toxin, which is released into the host cytosol following autoprocessing and inactivates small GTPases (PubMed:8810274, PubMed:16157585). Acts by mediating monoglycosylation of small GTPases of the Rho family (Rac1, RhoA, RhoG and Cdc42) in host cells at the conserved threonine residue located in the switch I region ('Thr-37/35'), using UDP-N-acetyl-alpha-D-glucosamine as the sugar donor (PubMed:8810274, PubMed:16157585). Monoglycosylation 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 (PubMed:8810274). {ECO:0000269|PubMed:16157585, ECO:0000269|PubMed:8810274}. |
| Temperature Dependency | |
| PH Dependency | |
| Pathway | |
| nucleotide Binding | |
| Features | Active site (2); Beta strand (7); Binding site (5); Chain (2); Domain (2); Helix (28); Metal binding (3); Mutagenesis (1); Region (10); Repeat (14); Site (1); Turn (5) |
| 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 |
| Interact With | |
| Induction | |
| Subcellular Location | SUBCELLULAR LOCATION: [Toxin A]: Secreted {ECO:0000250|UniProtKB:P18177}. Host endosome membrane {ECO:0000305|PubMed:20498856}. Note=Secreted from C.novyi cell into the extracellular environment via help of holin-like protein TcdE/UtxA (By similarity). 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 N-acetylglucosaminyltransferase TcdA form, which is released in the host cytosol (By similarity). {ECO:0000250|UniProtKB:P18177, ECO:0000269|PubMed:20498856}.; SUBCELLULAR LOCATION: [N-acetylglucosaminyltransferase 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 (N-acetylglucosaminyltransferase TcdA), which constitutes the active part of the toxin, in the host cytosol (PubMed:17334356). 1D-myo-inositol hexakisphosphate-binding (InsP6) activates the peptidase C80 domain and promotes autoprocessing (PubMed:17334356). {ECO:0000269|PubMed:17334356}. |
| Signal Peptide | |
| Structure 3D | X-ray crystallography (1) |
| Cross Reference PDB | 2VK9; |
| Mapped Pubmed ID | - |
| Motif | |
| Gene Encoded By | |
| Mass | 250,136 |
| Kinetics | BIOPHYSICOCHEMICAL PROPERTIES: [N-acetylglucosaminyltransferase TcdA]: Kinetic parameters: KM=307 uM for UDP-alpha-D-glucose {ECO:0000269|PubMed:16157585}; KM=17 uM for UDP-N-acetyl-alpha-D-glucosamine {ECO:0000269|PubMed:16157585}; Note=kcat is 282 h(-1) with UDP-alpha-D-glucose as substrate (PubMed:16157585). kcat is 2800 h(-1) with UDP-N-acetyl-alpha-D-glucosamine as substrate (PubMed:16157585). {ECO:0000269|PubMed:16157585}; |
| Metal Binding | METAL 284; /note=Magnesium or manganese; /evidence=ECO:0000250|UniProtKB:P18177; METAL 286; /note=Magnesium or manganese; /evidence=ECO:0000250|UniProtKB:P18177; METAL 520; /note=Magnesium or manganese; /evidence=ECO:0000250|UniProtKB:P18177 |
| Rhea ID | RHEA:64688; RHEA:64689 |
| Cross Reference Brenda | 2.4.1.B62; |