FIGURE 6.

rLytN functions as an amidase and endopeptidase. A, S. aureus wild-type peptidoglycan was treated with recombinant LytN for 16 h at 37 °C, reduced, and resolved by rpHPLC as described under “Experimental Procedures.” Individual peaks were desalted and subjected to MALDI-TOF MS, with observed m/z listed in red above each peak tested. Inset, Coomassie-Brilliant Blue-stained SDS-PAGE of rLytN. B, example mass spectra obtained from the three most prominent peaks shown in A (m/z 724.68, 796.69, and 2367.12), with the proposed structure for each compound (see Fig. 1 for color coding). C, diagram of cell wall anchored protein A (SpA). The cleavage sites of lysostaphin, mutanolysin, and LytN are noted with arrows. D, immunoblot with SpA-specific monoclonal antibody from protein lysates derived from staphylococci treated with lysostaphin, rLytN, or mutanolysin. mAU, milliabsorbance units.