Endopeptidase and glycosidase activities of the bacteriophage B30 lysin

Abstract

Synthetic peptides corresponding to portions of group B streptococcal peptidoglycan were used to show that the endopeptidase activity of bacteriophage B30 lysin cleaves between D-Ala in the stem peptide and L-Ala in the cross bridge and that the minimal peptide sequence cleaved is DL-gamma-Glu-Lys-D-Ala-Ala-Ala. The only glycosidase activity present is that of N-acetyl-beta-D-muramidase.

FIG. 1.

Domain structure of cloned bacteriophage B30 lysin (A) and structure of GBS peptidoglycan with sites of B30 lysin cleavage indicated (B). SH3b, putative bacterial cell wall-binding domain; His₆, hexahistidine affinity tag. The highlighted area depicts the minimum peptide structure required for endopeptidase cleavage.

FIG. 2.

Digestion of Ala-d-γ-Glu-Lys-d-Ala-Ala-Ala by phage B30 endopeptidase and analysis of products by capillary electrophoresis at pH 9.28. (A) The digest contains two peptides, revealed by their absorbance levels at 200 nm. They are Ala-d-γ-Glu-Lys-d-Ala (II) and Ala-Ala (III). (B) Digest supplemented with authentic Ala-d-γ-Glu-Lys-d-Ala-Ala-Ala (I). AU, absorbance units.

FIG. 3.

Gas chromatographic separation of the reduction products of a 2-h phage B30 lysin digest of GBS cell walls. It can be seen that after 2 h of digestion, most of the N-acetylmuramic acid present could be reduced with borohydride. Details of the analytical procedure are described in the text.