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PMID:19493008
| Citation |
Becker, SC, Dong, S, Baker, JR, Foster-Frey, J, Pritchard, DG and Donovan, DM (2009) LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells. FEMS Microbiol. Lett. 294:52-60 |
|---|---|
| Abstract |
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 active on purified peptidoglycan by positive-ion electrospray ionization MS. 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. 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. In contrast, truncations harboring just the amidase lytic domain show faint activity in both the zymogram and turbidity reduction assays, but no detectable activity in either plate lysis or MIC assays. 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. Both LysK and the CHAP-SH3b fusion were shown to lyse untreated S. aureus and the coagulase-negative strains. In the checkerboard assay, the CHAP-SH3b fusion achieves the same level of antimicrobial synergy with lysostaphin as the full-length LysK. |
| Links |
PubMed Online version:10.1111/j.1574-6968.2009.01541.x |
| Keywords |
Anti-Bacterial Agents/metabolism; Bacteriolysis; Endopeptidases/genetics; Endopeptidases/metabolism; Lysostaphin/metabolism; Microbial Sensitivity Tests; Microbial Viability; Sequence Deletion; Staphylococcus Phages/enzymology; Staphylococcus aureus/drug effects; Staphylococcus aureus/virology; Viral Proteins/genetics; Viral Proteins/metabolism |
| edit table |
Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
|
enables |
GO:0008745: N-acetylmuramoyl-L-alanine amidase activity |
ECO:0000314: direct assay evidence used in manual assertion |
F |
Seeded From UniProt |
complete | |||
|
involved_in |
GO:0001897: cytolysis by symbiont of host cells |
ECO:0000314: direct assay evidence used in manual assertion |
P |
Seeded From UniProt |
complete | |||
| GO:0008745: N-acetylmuramoyl-L-alanine amidase activity |
ECO:0000314: |
F |
Figure 1 shows the EIMS analysis of products of LysK reacting with S.aureus PG. Frame B of figure 1 shows where the enzyme must cut to form the product shown in frame A, between the NAM and the L-ala. |
complete | ||||
| GO:0001897: cytolysis by symbiont of host cells |
ECO:0000314: |
P |
Figure 5 shows LysK is able to lyse various Staph species. |
complete | ||||
| GO:0008233: peptidase activity |
ECO:0000315: |
F |
Figure 3: A series of LysK deletion constructs were tested using turbidity reduction assays. Constructs lacking the SH3B domain, LysK 211 and 390 showed 10-fold less activity then wild-type LysK. SH3B domains alone, 325-495 showed no discernable activity. 10μL spots containing 10μg of LysK297, or LysK149-495 had no discernable activity in the plate lysis assay. Fusion of SH3B to the CHAP domain, LysK221-390 showed increased activity of the CHAP domain. |
complete | ||||
See also
References
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