Gene Ontology annotation through association of InterPro records with GO terms

Gene Ontology annotation based on UniProtKB/Swiss-Prot keyword mapping

Electronic Gene Ontology annotations created by ARBA machine learning models

Combined Automated Annotation using Multiple IEA Methods

PMID:16199588

The recombinant phage lysin LysK has a broad spectrum of lytic activity against clinically relevant staphylococci, including methicillin-resistant Staphylococcus aureus.

Recombinant LysK expressed in Lactococcus lactis is enzymatically active against staphylococcal cell walls, giving a 54 kDa lytic zone on a zymogram containing MRSA strain DPC5645 that matches the predicted mass of LysK and is absent from vector controls.

"These results confirmed that recombinant LysK from lactococci is enzymatically active and capable of degrading staphylococcal cell walls."
LysK kills live pathogenic staphylococci, reducing MRSA numbers 99% within one hour in a kill curve.

"In kill curves using a human MRSA strain (DPC5645), a 99% reduction in staphylococcal cell numbers was observed 1 h after the addition of lysates containing LysK (Fig. 1B ), demonstrating that recombinant LysK is capable of killing live pathogenic staphylococci."
Lytic activity is restricted to the genus Staphylococcus; other Gram-positive genera, including probiotic lactobacilli, are unaffected.

"The lysin-containing lactococcal extract was incapable of lysing other gram-positive bacteria, such as Listeria innocua, Bacillus cereus, Lactobacillus rhamnosus , and Lactobacillus paracasei ."
The lysK transcript appears late in infection, between 10 and 20 minutes, consistent with a role at the lytic end of the cycle; the supporting data are not shown in the paper.

"Reverse transcription-PCR results demonstrated that the lysK transcript appears between 10 and 20 min after phage infection (data not shown)."

PMID:19047377

Phage lysin LysK can be truncated to its CHAP domain and retain lytic activity against live antibiotic-resistant staphylococci.

An independent group confirms that the CHAP domain alone is sufficient to lyse live clinical staphylococcal isolates including MRSA.

"A truncated derivative of the phage endolysin LysK containing only the CHAP (cysteine- and histidine-dependent amidohydrolase/peptidase) domain exhibited lytic activity against live clinical staphylococcal isolates, including methicillin-resistant Staphylococcus aureus."

PMID:19493008

LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells.

LysK is a three-domain endolysin: an N-terminal CHAP endopeptidase domain, a central amidase 2 domain, and a C-terminal SH3b cell wall-binding domain.

"LysK is a staphylococcal bacteriophage endolysin composed of three domains: an N-terminal cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) endopeptidase domain, a midprotein amidase 2 domain, and a C-terminal SH3b_5 (SH3b) cell wall-binding domain."
Both catalytic domains are directly shown to act on purified peptidoglycan by positive-ion electrospray ionisation mass spectrometry.

"Both catalytic domains are active on purified peptidoglycan by positive-ion electrospray ionization MS."
The two cleaved bonds are identified: the CHAP domain cuts between D-alanine of the stem peptide and glycine of the cross-bridge, and the amidase domain has N-acetylmuramoyl-L-alanine amidase activity, the same cut sites as the phi11 endolysin LytA.

"The cut sites are identical to LytA (phi11 endolysin), with cleavage between d-alanine of the stem peptide and glycine of the cross-bridge peptide, and N-acetylmuramoyl-l-alanine amidase activity."
Domain dissection shows the CHAP endopeptidase, not the amidase, carries the activity against live cells: CHAP-only truncations lyse S. aureus in zymogram, plate lysis and turbidity assays, while amidase-only truncations are barely active.

"Truncations of the LysK containing just the CHAP domain lyse Staphylococcus aureus cells in zymogram analysis, plate lysis, and turbidity reduction assays but have no detectable activity in a minimal inhibitory concentration (MIC) assay."
The C-terminal SH3b domain is needed for full activity: fusing it to the CHAP domain restores near full-length lytic activity.

"A fusion of the CHAP domain to the SH3b domain has near full-length LysK lytic activity, suggesting the need for a C-terminal binding domain."
Full-length LysK and the CHAP-SH3b fusion lyse untreated S. aureus and coagulase-negative staphylococci.

"Both LysK and the CHAP-SH3b fusion were shown to lyse untreated S. aureus and the coagulase-negative strains."

PMID:25064136

Crystal structure of the lytic CHAP(K) domain of the endolysin LysK from Staphylococcus aureus bacteriophage K.

The 1.69-1.79 A structures of the CHAP(K) domain (PDB 4CSH, 4CT3) reveal a papain-like fold with a catalytic triad in a hydrophobic cleft, placing the domain in the cysteine-protease mechanistic class.

"The structure of CHAP(K) revealed a papain-like topology with a hydrophobic cleft, where the catalytic triad is located."
A structural calcium ion is bound in an extra N-terminal lobe, coordinated by Asp45, Asp47, Tyr49, His51 and Asp56.

"CHAP(K) contains an additional lobe in its N-terminal domain, with a structural calcium ion, coordinated by residues Asp45, Asp47, Tyr49, His51 and Asp56."
A zinc ion sits in the active site next to the catalytic Cys54, but is loosely bound and its role is unresolved.

"The presence of a zinc ion in the active site was also apparent, coordinated by the catalytic residue Cys54 and a possible substrate analogue."
Alanine substitution of three of the calcium ligands abolishes staphylolytic activity, showing the calcium site is functionally required rather than incidental.

"Zymogram analysis demonstrated that mutation of residues Asp45, Asp47 and Asp56 resulted in the complete abolishment of the staphylolytic activity of the enzyme"
Cys54 and His117 are individually essential for activity and Glu134 strongly contributes, confirming the Cys-His-Glu catalytic triad.

"Mutation of the conserved Cys54 and His117 residues to alanine resulted in complete elimination of staphylolytic activity of the enzyme as demonstrated by zymographic analysis, indicating an essential role of these residues and supporting the hypothesis that they are part of the catalytic triad."
The division of labour between the two catalytic domains is restated: the amidase cuts MurNAc to L-alanine, the CHAP domain cuts D-alanine to the first glycine of the pentaglycine cross-bridge.

"The LysK amidase domain cleaves peptidoglycan between N-acetylmuramic acid and L-alanine of the stem peptide, while the CHAP domain hydrolyzes it between the D-alanine of the tetra-peptide stem and the first glycine of the penta-glycine cross-bridge"