| IED ID | IndEnz0002009016 |
| Enzyme Type ID | protease009016 |
| Protein Name |
Subtilase cytotoxin subunit A EC 3.4.21.- |
| Gene Name | subA |
| Organism | Escherichia coli |
| Taxonomic Lineage | cellular organisms Bacteria Proteobacteria Gammaproteobacteria Enterobacterales Enterobacteriaceae Escherichia Escherichia coli |
| Enzyme Sequence | MLKILWTYILFLLFISASARAEKPWYFDAIGLTETTMSLTDKNTPVVVSVVDSGVAFIGGLSDSEFAKFSFTQDGSPFPVKKSEALYIHGTAMASLIASRYGIYGVYPHALISSRRVIPDGVQDSWIRAIESIMSNVFLAPGEEKIINISGGQKGVASASVWTELLSRMGRNNDRLIVAAVGNDGADIRKLSAQQRIWPAAYHPVSSVNKKQDPVIRVAALAQYRKGETPVLHGGGITGSRFGNNWVDIAAPGQNITFLRPDAKTGTGSGTSEATAIVSGVLAAMTSCNPRATATELKRTLLESADKYPSLVDKVTEGRVLNAEKAISMFCKKNYIPVRQGRMSEEL |
| Enzyme Length | 347 |
| Uniprot Accession Number | Q6EZC2 |
| Absorption | |
| Active Site | ACT_SITE 52; /note="Charge relay system"; /evidence="ECO:0000255|PROSITE-ProRule:PRU01240, ECO:0000303|PubMed:15226357"; ACT_SITE 89; /note="Charge relay system"; /evidence="ECO:0000255|PROSITE-ProRule:PRU01240, ECO:0000303|PubMed:15226357"; ACT_SITE 272; /note="Charge relay system"; /evidence="ECO:0000255|PROSITE-ProRule:PRU01240, ECO:0000303|PubMed:15226357" |
| Activity Regulation | |
| Binding Site | |
| Calcium Binding | |
| catalytic Activity | |
| DNA Binding | |
| EC Number | 3.4.21.- |
| Enzyme Function | FUNCTION: Protease subunit of subtilase cytotoxin SubAB5 (PubMed:17024087). An endoprotease specific for host endoplasmic reticulum (ER) chaperone BiP/HSPA5, has no activity on human HSP70 or HSPA8 (PubMed:17024087). Cleaves between 'Leu-416' and 'Leu-417' of BiP/HSPA5 in the hinge between BiP's ATPase and protein-binding domains (PubMed:17024087). This induces host ER stress response and eventual cell death (PubMed:18005237, PubMed:18433465). Culture supernatant of E.coli expressing both subA and subB are toxic for Vero cells (African green monkey kidney cell line), Chinese hamster ovary cells and Hct-8 cells (human colonic epithelial cell line); the subunits are not toxic individually (PubMed:15226357). Purified SubAB5 is highly toxic, <0.1 pg is able to kill at least 50% of 30'000 Vero cells in a microtiter plate assay after 3 days; no cytotoxicity is seen at 24 hours (PubMed:15226357). Preabsorption with cells expressing a ganglioside GM2 mimic reduced cytotoxicity of SubAB5 by 93% in the Vero cytotoxicity assay (PubMed:15226357). Intraperitoneal injection of 200 ng of purified SubAB5 kills mice; the higher the dose the faster the mice die. Animals injected intraperitoneally with purified SubAB5 have microvascular thrombi in the brain and other organs, including the renal tubules and glomeruli (PubMed:15226357). Injection induces an unfolded response in mice (PubMed:17024087). Mice fed E.coli cells expressing cloned SubAB5 experience drastic weight loss and appear ill and lethargic (PubMed:15226357). Protein synthesis in Vero cells is transiently inhibited by SubAB5; both subunits are required for this effect (PubMed:17101670, PubMed:18005237, PubMed:18433465). Inhibition of protein synthesis is prevented by brefeldin A; cells are arrested in the G1 phase (PubMed:18005237). SubAB5 at 100 ng/ml induced caspase-dependent apoptosis in Vero cells through mitochondrial membrane damage (PubMed:19380466). {ECO:0000269|PubMed:15226357, ECO:0000269|PubMed:17024087, ECO:0000269|PubMed:17101670, ECO:0000269|PubMed:18005237, ECO:0000269|PubMed:18433465, ECO:0000269|PubMed:19380466}. |
| Temperature Dependency | |
| PH Dependency | |
| Pathway | |
| nucleotide Binding | |
| Features | Active site (3); Beta strand (14); Chain (1); Disulfide bond (1); Domain (1); Helix (13); Motif (1); Mutagenesis (1); Natural variant (1); Region (1); Signal peptide (1); Turn (5) |
| Keywords | 3D-structure;Direct protein sequencing;Disulfide bond;Host cytoplasm;Host endoplasmic reticulum;Hydrolase;Plasmid;Protease;Secreted;Serine protease;Signal;Toxin;Virulence |
| Interact With | |
| Induction | INDUCTION: Part of the subA-subB operon (PubMed:15226357). {ECO:0000269|PubMed:15226357}. |
| Subcellular Location | SUBCELLULAR LOCATION: Secreted {ECO:0000269|PubMed:15226357}. Host cytoplasm, host cytosol {ECO:0000269|PubMed:15226357, ECO:0000269|PubMed:17101670}. Host endoplasmic reticulum lumen {ECO:0000305|PubMed:17024087, ECO:0000305|PubMed:18005237, ECO:0000305|PubMed:23921389}. Note=Colocalizes with host BiP/HSPA5 in the endoplasmic reticulum of Vero cells, its activity on BiP/HSPA5 is blocked by pretreatment with brefeldin A, which disrupts the Golgi apparatus and inhibits retrograde transport from the cell surface to the Golgi (PubMed:17024087, PubMed:18005237). Using different inhibitors it has been shown to be actively internalized by membrane-bound vesicles and undergoes clathrin-dependent retrograde transport, via early endosomes and the Golgi network, to the endoplasmic reticulum (PubMed:18042253). Trafficking is similar in Vero cells, human HeLa cells and murine N2A cells (PubMed:18042253). {ECO:0000269|PubMed:15226357, ECO:0000269|PubMed:17024087, ECO:0000269|PubMed:18005237, ECO:0000269|PubMed:18042253}. |
| Modified Residue | |
| Post Translational Modification | |
| Signal Peptide | SIGNAL 1..21; /evidence="ECO:0000255, ECO:0000305|PubMed:15226357" |
| Structure 3D | X-ray crystallography (2) |
| Cross Reference PDB | 2IY9; 4BWG; |
| Mapped Pubmed ID | - |
| Motif | MOTIF 344..347; /note=Prevents secretion from ER; /evidence=ECO:0000255 |
| Gene Encoded By | Plasmid megaplasmid pO113 |
| Mass | 37,494 |
| Kinetics | |
| Metal Binding | |
| Rhea ID | |
| Cross Reference Brenda |