Subtilisin E (aprE) is one of the major secreted alkaline serine proteases of Bacillus subtilis, belonging to the peptidase S8 (subtilisin) family. The enzyme is synthesized as a preproenzyme with an N-terminal signal peptide (residues 1-29) that directs secretion via the Sec pathway, and a propeptide (residues 30-106) that functions as an intramolecular chaperone essential for proper folding of the mature protease domain. The propeptide is autocatalytically cleaved after folding is complete. The mature enzyme (residues 107-381) contains the canonical Asp-His-Ser catalytic triad (Asp138, His170, Ser327) characteristic of subtilisins and requires calcium ions for structural stability (two Ca2+ binding sites per subunit). Subtilisin E exhibits broad substrate specificity, preferentially cleaving after large uncharged residues, and is inhibited by serine protease inhibitors such as PMSF but not by metalloprotease inhibitors like EDTA. Beyond its role in extracellular protein degradation for nutrient acquisition, aprE participates in quorum sensing by processing the Phr family of signaling peptide precursors (including proCSF/PhrC and proPhrA) to generate active pentapeptide pheromones that regulate competence development and sporulation. Expression is induced during stationary phase under control of the DegS-DegU two-component system and modulated by global regulators including AbrB, SinR, and ScoC. Although subtilisin secretion is associated with the onset of sporulation, the enzyme itself is not required for normal sporulation. Subtilisin E is one of the most industrially important enzymes, widely used in detergent formulations and various biotechnological applications.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004252 serine-type endopeptidase activity | IBA GO_REF:0000033 | ACCEPT | Summary: IBA annotation for serine-type endopeptidase activity is well-supported. Subtilisin E is a canonical member of the peptidase S8 family with the conserved catalytic triad (Asp138-His170-Ser327) characteristic of serine endopeptidases. The enzyme hydrolyzes proteins with broad specificity, preferring large uncharged residues at P1 (UniProt P04189). Reason: This is the core molecular function of Subtilisin E. The enzyme contains the canonical Asp-His-Ser catalytic triad of the S8 subtilisin family, confirmed by X-ray crystallography (PMID:9811547). Biochemical studies demonstrate broad endopeptidase activity with preference for hydrophobic residues, inhibited by PMSF but not EDTA. This is the most specific and accurate term for the enzymatic activity. Supporting Evidence: UniProt:P04189 An extracellular alkaline serine protease, it catalyzes the hydrolysis of proteins and peptide amides PMID:9811547 The crystal structure of an autoprocessed Ser221Cys-subtilisin E-propeptide complex at 2.0 A resolution |
| GO:0006508 proteolysis | IBA GO_REF:0000033 | ACCEPT | Summary: IBA annotation for proteolysis is appropriate as a biological process term corresponding to the serine-type endopeptidase molecular function. Subtilisin E functions in extracellular protein degradation for nutrient acquisition and in processing signaling peptide precursors. Reason: Proteolysis is the appropriate biological process term for a secreted protease. The enzyme performs extracellular protein degradation and also specifically processes Phr signaling peptide precursors (PMID:17666034). This general term appropriately captures the biological process. Supporting Evidence: UniProt:P04189 An extracellular alkaline serine protease, it catalyzes the hydrolysis of proteins and peptide amides PMID:17666034 Purified subtilisin and Vpr were shown to be capable of processing proCSF as well as at least one other Phr peptide produced by B |
| GO:0004252 serine-type endopeptidase activity | IEA GO_REF:0000120 | ACCEPT | Summary: IEA annotation redundant with the IBA annotation for the same term. The annotation is correct based on EC number mapping (EC 3.4.21.62). Reason: This IEA annotation is derived from the EC number (EC 3.4.21.62) assigned to Subtilisin E. While redundant with the IBA annotation, it provides independent evidence supporting the core molecular function. The term is appropriate and accurate. Supporting Evidence: UniProt:P04189 Hydrolysis of proteins with broad specificity for peptide bonds, and a preference for a large uncharged residue in P1. Hydrolyzes peptide amides.; EC=3.4.21.62 |
| GO:0005576 extracellular region | IEA GO_REF:0000044 | ACCEPT | Summary: IEA annotation for extracellular region is well-supported by experimental evidence. Subtilisin E is synthesized with an N-terminal signal peptide (residues 1-29) and is secreted via the Sec pathway to function in the extracellular environment (UniProt P04189; PMID:3090033). Reason: This is the correct cellular component term for Subtilisin E. The enzyme is secreted via the Sec pathway after signal peptide cleavage and functions extracellularly. The signal peptide cleavage site has been determined (PMID:3090033). Supporting Evidence: UniProt:P04189 SUBCELLULAR LOCATION: Secreted PMID:3090033 The preprosubtilisin was found to have a 29-amino-acid-long signal peptide with the signal peptidase cleavage sequence of AlaGln-AlaAla |
| GO:0006508 proteolysis | IEA GO_REF:0000120 | ACCEPT | Summary: IEA annotation for proteolysis is redundant with the IBA annotation for the same term. The annotation is appropriate. Reason: While redundant with the IBA annotation, this IEA provides independent evidence support for the proteolysis annotation based on keyword mapping. The term is appropriate for a secreted protease. Supporting Evidence: UniProt:P04189 An extracellular alkaline serine protease, it catalyzes the hydrolysis of proteins and peptide amides |
| GO:0008233 peptidase activity | IEA GO_REF:0000120 | MARK AS OVER ANNOTATED | Summary: IEA annotation for peptidase activity is a parent term of the more specific serine-type endopeptidase activity (GO:0004252). This represents a less informative annotation. Reason: While technically correct, this is a high-level parent term of the more specific serine-type endopeptidase activity (GO:0004252) already annotated. The more specific term is preferred as it provides more information about the enzymatic mechanism. This annotation does not add informational value beyond what is captured by GO:0004252. Supporting Evidence: UniProt:P04189 Hydrolysis of proteins with broad specificity for peptide bonds, and a preference for a large uncharged residue in P1. Hydrolyzes peptide amides.; EC=3.4.21.62 |
| GO:0008236 serine-type peptidase activity | IEA GO_REF:0000120 | MARK AS OVER ANNOTATED | Summary: IEA annotation for serine-type peptidase activity is a direct parent of serine-type endopeptidase activity (GO:0004252), which is already annotated at the most specific level. Analogous to peptidase activity (GO:0008233), which is MARK_AS_OVER_ANNOTATED for the same reason. Reason: GO:0008236 (serine-type peptidase activity) is a strict parent of GO:0004252 (serine-type endopeptidase activity), which is already annotated. There is no principled basis to accept GO:0008236 while marking the sibling parent GO:0008233 (peptidase activity) as over-annotated; both are fully subsumed by GO:0004252. Consistent treatment with GO:0008233 requires MARK_AS_OVER_ANNOTATED. Supporting Evidence: UniProt:P04189 Inhibited by PMSF (phenylmethylsulphonyl fluoride) and 3,4-dichloroisocoumarin but not by EDTA |
| GO:0016787 hydrolase activity | IEA GO_REF:0000043 | MARK AS OVER ANNOTATED | Summary: IEA annotation for hydrolase activity is a very general parent term. Subtilisin E is specifically a serine-type endopeptidase, making this annotation overly broad. Reason: This is a very high-level term in the GO hierarchy. While technically correct (proteases are hydrolases), this term provides minimal information about the specific function of Subtilisin E. The more specific term GO:0004252 (serine-type endopeptidase activity) is far more informative and already annotated. This annotation does not add value. Supporting Evidence: UniProt:P04189 Hydrolysis of proteins with broad specificity for peptide bonds, and a preference for a large uncharged residue in P1. Hydrolyzes peptide amides.; EC=3.4.21.62 |
| GO:0030435 sporulation resulting in formation of a cellular spore | IEA GO_REF:0000043 | REMOVE | Summary: IEA annotation for sporulation is questionable. While aprE expression is temporally associated with sporulation onset, experimental evidence shows that subtilisin is NOT required for normal sporulation. Disruption mutants show wild-type sporulation (PMID:6427178). Reason: This annotation is based on the UniProt keyword Sporulation, but the experimental evidence contradicts a direct role in sporulation. The disruption phenotype study (PMID:6427178) explicitly states that aprE mutants show wild-type sporulation. The UniProt entry also notes that subtilisin is not necessary for normal sporulation. The temporal association with sporulation onset reflects shared regulation (via Spo0A/AbrB pathways) rather than functional involvement in spore formation. Supporting Evidence: PMID:6427178 Physiological characterization of the delta apr-684 mutation revealed no discernable effect on the formation of heat-resistant endospores, but strains carrying the mutation produced only 10% of wild-type serine protease activity UniProt:P04189 subtilisin is not necessary for normal sporulation |
| GO:0046872 metal ion binding | IEA GO_REF:0000043 | MODIFY | Summary: IEA annotation for metal ion binding is supported by structural evidence. Subtilisin E binds two calcium ions per subunit that are essential for structural stability. However, calcium ion binding (GO:0005509) would be more specific. Reason: Subtilisin E specifically binds calcium ions (Ca2+), not general metal ions. X-ray crystallography (PMID:9811547) confirms two calcium binding sites per subunit with specific coordinating residues identified. The more specific term GO:0005509 (calcium ion binding) would better represent this function. Proposed replacements: calcium ion binding Supporting Evidence: UniProt:P04189 Name=Ca(2+); Xref=ChEBI:CHEBI:29108; Evidence={ECO:0000269|PubMed:9811547}; Note=Binds 2 calcium ions per subunit |
| GO:0008236 serine-type peptidase activity | IDA PMID:17666034 Identification of subtilisin, Epr and Vpr as enzymes that pr... | MODIFY | Summary: IDA annotation for serine-type peptidase activity based on PMID:17666034, which demonstrated endoproteolytic cleavage of proCSF to the active CSF pentapeptide. The same evidence supports the more specific GO:0004252 (serine-type endopeptidase activity), as proCSF-to-CSF processing is an internal-bond cleavage event. Reason: The proCSF-to-CSF processing in PMID:17666034 is endoproteolytic (internal peptide bond cleavage to generate the mature pentapeptide). This directly supports GO:0004252 (serine-type endopeptidase activity) rather than the less specific parent GO:0008236. Upgrading retains the IDA experimental evidence on the most informative term. Proposed replacements: serine-type endopeptidase activity Supporting Evidence: PMID:17666034 Using both a cellular and a mass spectrometric approach, we determined that a sigma-H-regulated, secreted, serine protease(s) cleaved proCSF to CSF |
| GO:0009274 peptidoglycan-based cell wall | IDA PMID:17666034 Identification of subtilisin, Epr and Vpr as enzymes that pr... | REMOVE | Summary: IDA annotation for peptidoglycan-based cell wall localization appears to be an error. PMID:17666034 studied the role of subtilisin in CSF production but does not provide evidence for cell wall localization. Subtilisin E is a secreted extracellular enzyme. Reason: This annotation appears to be erroneous. The cited paper (PMID:17666034) does not provide evidence for cell wall localization of subtilisin. The study focused on identifying proteases that process CSF signaling peptides. All available evidence indicates Subtilisin E is secreted to the extracellular region, not localized to the cell wall. The appropriate cellular component is GO:0005576 (extracellular region). Supporting Evidence: UniProt:P04189 SUBCELLULAR LOCATION: Secreted PMID:17666034 a sigma-H-regulated, secreted, serine protease(s) cleaved proCSF to CSF |
| GO:0009372 quorum sensing | IDA PMID:17666034 Identification of subtilisin, Epr and Vpr as enzymes that pr... | ACCEPT | Summary: IDA annotation for quorum sensing is well-supported by PMID:17666034. The study demonstrates that subtilisin processes proCSF to produce the mature CSF pentapeptide, which functions as a cell-cell signaling molecule in quorum sensing pathways regulating competence and sporulation. Reason: PMID:17666034 provides direct experimental evidence that subtilisin participates in quorum sensing by processing the proCSF precursor to generate the active CSF (PhrC) signaling pentapeptide. CSF is a key quorum sensing molecule in B. subtilis that regulates competence development and sporulation initiation through the Rap-Phr system. This represents a specific biological role beyond general proteolysis. Supporting Evidence: PMID:17666034 Cell-cell communication regulates many important processes in bacteria PMID:17666034 Gram-positive bacteria use peptide signals for communication, such as the Phr pentapeptides of Bacillus subtilis |
| GO:0140448 signaling receptor ligand precursor processing | IDA PMID:17666034 Identification of subtilisin, Epr and Vpr as enzymes that pr... | ACCEPT | Summary: IDA annotation for signaling receptor ligand precursor processing is strongly supported by PMID:17666034. The study directly demonstrates that subtilisin processes the proCSF and proPhrA precursors to generate active signaling pentapeptides. Reason: This is an excellent and specific annotation capturing a key biological function of subtilisin. PMID:17666034 provides direct evidence that subtilisin cleaves proCSF and proPhrA precursors to generate the mature signaling peptides. The Phr peptides function as ligands for the Rap phosphatases, making this term highly appropriate. Supporting Evidence: PMID:17666034 The Phr pentapeptides are secreted with a pro domain that is cleaved to produce an active signalling peptide |
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