PMID:23733182

Citation	Regulski, K, Courtin, P, Kulakauskas, S and Chapot-Chartier, MP (2013) A novel type of peptidoglycan-binding domain highly specific for amidated D-Asp cross-bridge, identified in Lactobacillus casei bacteriophage endolysins. J. Biol. Chem. 288:20416-26
Abstract	Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-L-alanine amidase, whereas Lc-Lys-2 is a γ-D-glutamyl-L-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with D-Ala(4)→D-Asx-L-Lys(3) in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting D-Ala(4)→L-Ala-(L-Ala/L-Ser)-L-Lys(3); moreover, they do not lyse the L. lactis mutant containing only the nonamidated D-Asp cross-bridge, i.e. D-Ala(4)→D-Asp-L-Lys(3). In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 L-Lys(3)-D-Asn-L-Lys(3) bridges replacing the wild-type 4→3 D-Ala(4)-D-Asn-L-Lys(3) bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly D-Asn but not PG with only the nonamidated D-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the D-Asn interpeptide bridge of PG.
Links	PubMed PMC3711307 Online version:10.1074/jbc.M112.446344
Keywords	Amides/metabolism; Amino Acid Sequence; Asparagine/genetics; Asparagine/metabolism; Aspartic Acid/genetics; Aspartic Acid/metabolism; Bacteriophages/enzymology; Bacteriophages/genetics; Binding Sites/genetics; Catalytic Domain/genetics; Cell Wall/metabolism; Electrophoresis, Polyacrylamide Gel; Endopeptidases/genetics; Endopeptidases/metabolism; Gram-Positive Bacteria/genetics; Gram-Positive Bacteria/metabolism; Lactobacillus casei/enzymology; Lactobacillus casei/genetics; Lactobacillus casei/virology; Microscopy, Fluorescence; Molecular Sequence Data; Mutation; N-Acetylmuramoyl-L-alanine Amidase/genetics; N-Acetylmuramoyl-L-alanine Amidase/metabolism; Peptidoglycan/metabolism; Prophages/enzymology; Prophages/genetics; Protein Binding; Sequence Homology, Amino Acid; Substrate Specificity
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
LACCA:A0A0E2LWA2		GO:0004175: endopeptidase activity	ECO:0000314:		F		fig. 3B and Table 3 show the hydrolytic specificity of the recombinant pure Lc-Lys-2 on Lactobacillus casei-purified Peptidoglycan (PG). The PG fragment obtained by digestion with Lc-Lys-2 was separated by RP-HPLC and analyzed by MALDI-TOF mass spectrometry. The HPLC profile reveal the hydrolytic specificities for Lc-Lys-2. MS analysis of the major peaks and comparison of the obtained masses with the reference L. casei PG structure enabled identification of peptides generated by Lc-Lys-2 (Table 3) and to deduce the cleavage specificity (Fig. 3D). Lc-Lys-2 has a gamma-D-glutamyl-L-lysyl endopeptidase specificity.	complete CACAO 12620
LACCA:K0N732		GO:0008745: N-acetylmuramoyl-L-alanine amidase activity	ECO:0000314:		F		Fig. 3B and Table 3 show the hydrolytic specificity of recombinant pure Lc-Lys on L. casei-purified Peptidoglycan (PG). The PG fragments obtained by digestion with Lc-Lys was separated by RP-HPLC and analyzed by MALDI-TOF mass spectrometry. the HPLC profile reveal the hydrolytic specificities for Lc-Lys. MS analysis of the major peaks and comparison of the obtained masses with the reference L. casei PG structure enabled identification of peptides generated by Lc-Lys (Table 3) and to deduce the cleavage specificity (Fig. 3D). Lc-Lys has an N-acetylmuramoyl-L-alanine amidase specificity,	complete CACAO 12621
LACCA:K0N3B2		GO:0004175: endopeptidase activity	ECO:0000314:		F		Fig. 3B and Table 3 show the hydrolytic specificity of recombinant pure Lc-Lys-2 on L. casei-purified Peptidoglycan (PG). The PG fragments obtained by digestion with Lc-Lys-2 was separated by RP-HPLC and analyzed by MALDI-TOF mass spectrometry. The HPLC profile reveal the hydrolytic specificities for Lc-Lys-2. MS analysis of the major peaks and comparison of the obtained masses with the reference L. casei PG structure enabled identification of peptides generated by Lc-Lys-2 (Table 3) and to deduce the cleavage specificity (Fig. 3D). Lc-Lys-2 has a gamma-D-glutamyl-L-lysyl endopeptidase specificity.	complete CACAO 12622
LACCD:A0A158SHI5		GO:0008745: N-acetylmuramoyl-L-alanine amidase activity	ECO:0000314:		F		Fig. 3B and Table 3 show the hydrolytic specificity of recombinant pure Lc-Lys on L. casei-purified Peptidoglycan (PG). The PG fragments obtained by digestion with Lc-Lys was separated by RP-HPLC and analyzed by MALDI-TOF mass spectrometry. the HPLC profile reveal the hydrolytic specificities for Lc-Lys. MS analysis of the major peaks and comparison of the obtained masses with the reference L. casei PG structure enabled identification of peptides generated by Lc-Lys (Table 3) and to deduce the cleavage specificity (Fig. 3D). Lc-Lys has an N-acetylmuramoyl-L-alanine amidase specificity,	complete CACAO 12623
LACCD:A0A125U8R1		GO:0004175: endopeptidase activity	ECO:0000314:		F		Fig. 3B and Table 3 show the hydrolytic specificity of recombinant pure Lc-Lys-2 on L. casei-purified Peptidoglycan (PG). The PG fragments obtained by digestion with Lc-Lys-2 was separated by RP-HPLC and analyzed by MALDI-TOF mass spectrometry. The HPLC profile reveal the hydrolytic specificities for Lc-Lys-2. MS analysis of the major peaks and comparison of the obtained masses with the reference L. casei PG structure enabled identification of peptides generated by Lc-Lys-2 (Table 3) and to deduce the cleavage specificity (Fig. 3D). Lc-Lys-2 has a gamma-D-glutamyl-L-lysyl endopeptidase specificity.	complete CACAO 12624

References

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