LytN is a secreted peptidoglycan (murein) hydrolase of Staphylococcus aureus that acts in the cross-wall (septal) compartment during cell division and envelope assembly. Its precursor carries a YSIRK-G/S signal peptide that directs secretion to the mid-cell cross-wall; it also has a LysM peptidoglycan-binding module and a catalytic CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain. The CHAP domain is bifunctional, acting as both an N-acetylmuramoyl-L-alanine amidase (cleaving the MurNAc-L-Ala amide bond linking the glycan strand to the stem peptide) and a D-alanyl-glycine endopeptidase (cleaving the D-Ala-Gly peptide bond); catalysis depends on the conserved CHAP residues Cys266 and His329. By hydrolyzing septal peptidoglycan, LytN promotes cross-wall splitting and the completion of daughter-cell separation. Its activity is spatially controlled (wall teichoic acid mediates its septal localization) and dosage-controlled: loss of LytN damages the cross-wall and impairs growth, while overexpression triggers cross-wall rupture and lysis. LytN also contributes to remodeling the peptidoglycan anchor of surface proteins such as protein A, removing amino sugars from the attached peptidoglycan, although it is not the determinant of overall protein A release.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005576 extracellular region | IEA GO_REF:0000044 | MARK AS OVER ANNOTATED | Summary: LytN is a secreted protein (UniProt SubCell "Secreted"), so the broad extracellular-region term is not incorrect. However, LytN does not act freely in the extracellular space; it is delivered via a YSIRK-G/S signal peptide to the cross-wall (division septum) where it carries out septal peptidoglycan hydrolysis, and its function depends on this localization. Reason: The SubCell-derived "extracellular region" is technically defensible but uninformative and partly misleading for a cross-wall enzyme. Frankel et al. showed externally added purified LytN does not complement a lytN mutant, whereas bacterially produced precursor does, demonstrating that physiological function requires cross-wall targeting rather than bulk extracellular activity (PMID:21784864). A cell septum / cell wall cellular-component term is more accurate (see NEW annotations); retaining this term as non-core/over-annotated is acceptable since the protein is genuinely secreted. Supporting Evidence: PMID:21784864 Secreted polypeptides endowed with a YSIRK-G/S signal peptide are directly delivered to the cross-wall compartment. One such YSIRK-containing protein is the murein hydrolase LytN. PMID:21784864 The lytN phenotype can be reversed by the controlled expression of lytN but not by adding purified LytN to staphylococcal cultures. |
| GO:0008932 lytic endotransglycosylase activity | IEA GO_REF:0000118 | REMOVE | Summary: This TreeGrafter/PANTHER annotation (from subfamily PTHR33734:SF22, "membrane-bound lytic murein transglycosylase D") is mechanistically incorrect for LytN. Lytic transglycosylases cleave the glycosidic MurNAc-GlcNAc bond non-hydrolytically, forming a 1,6-anhydro-MurNAc product (GO:0008932 definition). LytN has no transglycosylase domain; its catalytic module is a CHAP domain that HYDROLYZES amide and peptide bonds, acting as an N-acetylmuramoyl-L-alanine amidase and a D-alanyl-glycine endopeptidase. Reason: The annotation arises from an over-propagated subfamily misassignment. PANTHER places LytN in PTHR33734:SF22 (a lytic transglycosylase subfamily), but LytN's experimentally characterized activities are hydrolytic amide/peptide cleavages, not the 1,6-anhydro-forming glycosidic cleavage that defines lytic endotransglycosylase activity (GO:0008932, verified via QuickGO). LytN's domain architecture (CHAP + LysM, no transglycosylase fold) and the direct product evidence (MurNAc-L-Ala and D-Ala-Gly bond cleavage, abolished by Cys266/His329 mutation) contradict transglycosylase activity (PMID:21784864). This electronic inference should be removed and replaced by the verified amidase and endopeptidase MF terms (see NEW annotations). Supporting Evidence: PMID:21784864 LytN harbors LysM and CHAP domains, the latter of which functions as both an N-acetylmuramoyl-L-alanine amidase and D-alanyl-glycine endopeptidase. |
| GO:0016787 hydrolase activity | IEA GO_REF:0000104 | MODIFY | Summary: LytN is indeed a hydrolase (a peptidoglycan hydrolase / murein hydrolase), so this term is correct but uninformatively general. The specific activities are an N-acetylmuramoyl-L-alanine amidase and a peptidoglycan (D-alanyl-glycine) endopeptidase, both well supported experimentally. Reason: GO:0016787 is a root-level hydrolase term that conveys little about LytN's function. It should be replaced by the specific catalytic activities demonstrated by Frankel et al. (PMID:21784864), namely N-acetylmuramoyl-L-alanine amidase activity (GO:0008745) and peptidoglycan endopeptidase activity (GO:0061785). Both replacement terms were verified via QuickGO. Proposed replacements: N-acetylmuramoyl-L-alanine amidase activity peptidoglycan endopeptidase activity Supporting Evidence: PMID:21784864 LytN harbors LysM and CHAP domains, the latter of which functions as both an N-acetylmuramoyl-L-alanine amidase and D-alanyl-glycine endopeptidase. |
| GO:0008745 N-acetylmuramoyl-L-alanine amidase activity | IDA PMID:21784864 LytN, a murein hydrolase in the cross-wall compartment of St... | NEW | Summary: The CHAP domain of LytN cleaves the MurNAc-L-Ala amide bond, an N-acetylmuramoyl-L-alanine amidase activity demonstrated biochemically by muropeptide product analysis (MS) and Edman degradation, and abolished by the CHAP active-site mutations C266A and H329A. Reason: Direct experimental evidence supports this specific molecular function (PMID:21784864), the verified, informative replacement for the generic hydrolase term. GO:0008745 verified via QuickGO ("hydrolysis of the link between N-acetylmuramoyl residues and L-amino acid residues in bacterial cell-wall glycopeptides"). Supporting Evidence: PMID:21784864 LytN harbors LysM and CHAP domains, the latter of which functions as both an N-acetylmuramoyl-L-alanine amidase and D-alanyl-glycine endopeptidase. |
| GO:0061785 peptidoglycan endopeptidase activity | IDA PMID:21784864 LytN, a murein hydrolase in the cross-wall compartment of St... | NEW | Summary: LytN's CHAP domain also acts as a D-alanyl-glycine endopeptidase, cleaving the D-Ala-Gly peptide bond within the staphylococcal peptidoglycan stem/cross-bridge; this is a peptidoglycan endopeptidase activity. Reason: Direct biochemical evidence (product analysis and active-site mutagenesis) supports a peptidoglycan endopeptidase activity (D-Ala-Gly cleavage) for LytN (PMID:21784864), corroborated as a recognized D-Ala-Gly hydrolase (PMID:39495121). GO:0061785 verified via QuickGO ("an endopeptidase activity that uses peptidoglycan as a substrate"). Supporting Evidence: PMID:21784864 LytN harbors LysM and CHAP domains, the latter of which functions as both an N-acetylmuramoyl-L-alanine amidase and D-alanyl-glycine endopeptidase. PMID:39495121 The only other recognised D-Ala-Gly hydrolase in S. aureus is LytN. |
| GO:0009253 peptidoglycan catabolic process | IDA PMID:21784864 LytN, a murein hydrolase in the cross-wall compartment of St... | NEW | Summary: By hydrolyzing amide and peptide bonds in mature peptidoglycan, LytN participates in the breakdown/remodeling of the murein sacculus. Reason: LytN is a murein hydrolase that cleaves bonds in mature peptidoglycan (PMID:21784864); peptidoglycan catabolic process (GO:0009253, verified via QuickGO) captures this biological process. This is the BP counterpart of the hydrolytic MF terms. Supporting Evidence: PMID:21784864 One such YSIRK-containing protein is the murein hydrolase LytN. |
| GO:0000920 septum digestion after cytokinesis | IMP PMID:21784864 LytN, a murein hydrolase in the cross-wall compartment of St... | NEW | Summary: LytN promotes cross-wall (septal peptidoglycan) splitting and the completion of daughter-cell separation; lytN mutants show structural damage to the cross-wall and impaired growth, consistent with a role in septal cell wall digestion that enables daughter-cell separation. Reason: Frankel et al. demonstrate that LytN secretion into the cross-wall promotes peptidoglycan separation and completion of the cell cycle, and that its loss damages the cross-wall (PMID:21784864). GO:0000920 "septum digestion after cytokinesis" (verified via QuickGO) captures the enzymatic breakdown of septal cell wall enabling daughter-cell separation. Supporting Evidence: PMID:21784864 LytN secretion into the cross-wall promotes peptidoglycan separation and completion of the staphylococcal cell cycle. PMID:21784864 lytN mutations precipitate structural damage to the cross-wall and interfere with staphylococcal growth. |
| GO:0071555 cell wall organization | IMP PMID:21784864 LytN, a murein hydrolase in the cross-wall compartment of St... | NEW | Summary: LytN contributes to staphylococcal envelope assembly and cross-wall remodeling; its loss impairs proper growth and envelope assembly. This term is also asserted as a UniProt keyword-derived annotation in the UniProt entry. Reason: LytN remodels cell-wall peptidoglycan during division and envelope assembly (PMID:21784864); cell wall organization (GO:0071555, verified via QuickGO; also present as IEA UniProtKB-KW in the UniProt record) is an appropriate, well-supported biological process term. Supporting Evidence: PMID:21784864 is involved in proper bacterial growth and envelope assembly |
| GO:0030428 cell septum | IDA PMID:21784864 LytN, a murein hydrolase in the cross-wall compartment of St... | NEW | Summary: LytN is delivered to and acts in the cross-wall / division septum compartment at mid-cell, the location where septal peptidoglycan remodeling occurs. Reason: The YSIRK-G/S signal peptide targets LytN to the cross-wall (septal) compartment, where it functions; this is a more accurate cellular-component term than the broad extracellular region (PMID:21784864; WTA-mediated septal localization in PMID:35675307). GO:0030428 "cell septum" verified via QuickGO. Supporting Evidence: PMID:21784864 Secreted polypeptides endowed with a YSIRK-G/S signal peptide are directly delivered to the cross-wall compartment. PMID:35675307 WTA also mediates the specific binding of Sle1 and LytN to the cross-wall of the septum |
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Download this section (compressed HTML)Q: Are the amidase (MurNAc-L-Ala) and endopeptidase (D-Ala-Gly) activities of LytN both required in vivo for cross-wall splitting, or does one dominate the physiological function?
Q: How is LytN activity restricted to the cross-wall and prevented from digesting peripheral wall, given that overexpression causes lethal lysis? What are the relative contributions of the YSIRK-G/S signal peptide, the LysM domain, and wall teichoic acid to this spatial control?
Q: How does LytN functionally overlap or coordinate with the other S. aureus cross-wall hydrolases (Atl, Sle1, LytM) during daughter-cell separation and surface-protein anchor remodeling?
Experiment: Separation-of-function mutagenesis of the CHAP domain to uncouple the amidase and endopeptidase activities, followed by complementation of a lytN mutant, to determine which activity drives cross-wall splitting in vivo.
Experiment: Quantitative super-resolution or fluorescence imaging of tagged LytN in wild-type versus WTA-deficient (e.g. tarO) backgrounds to test the requirement of wall teichoic acid for septal localization and to time LytN recruitment relative to division.
Experiment: Defined in vitro digestion of purified staphylococcal peptidoglycan (and isolated muropeptides) with recombinant LytN and Cys266/His329 variants, with LC-MS muropeptide profiling, to map bond preferences and the dependence of amidase activity on glycan context.
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