FIG 5.

Single-substrate assays demonstrating the l,d-endopeptidase activity of LdcA enzymes on cross-linked muropeptides. (A) Two hydrolyzable bonds in s11 indicated with black and gray arrows correspond to routes “a” and “b,” respectively. (B to G) LC-MS traces of LdcA reactions with s11. (H to J) Mass spectra of s11 reaction products p2, p13, and p14. (K and L) Collision-induced dissociation (CID) MS/MS spectra of p13 and p13′ confirm hydrolysis of s11 by route “a.” The pink bar indicates the loss of Ala from the N terminus (71 Da), while the blue bar indicates the loss of Ala from the C terminus (89 Da). (M to Q) Reactions of LdcA with s12 (M) and the resulting substrate and product LC-MS traces (N to Q). (R to U) CID MS/MS spectra of detected route “a” products (p16 and p17) and potential route “b” products (p16′ and p17′). Further LC-MS/MS data are shown in Fig. S6.