Error bars represent the standard error of the mean. Figure learn more 4 SrtB ΔN26 substrate specificity. Purified recombinant SrtBΔN26 protein was incubated with a range of peptide sequences to investigate its substrate specificity. The motifs SPKTG, PPKTG, SPSTG and SPQTG were all recognized and cleaved following incubation with SrtBΔN26. The scrambled peptide sequences GSKTP, GPKTG, GSSTP, and GSQTP serve as controls for the cleavage specificity of SrtBΔN26. The sequences LPETG

and NPQTN, corresponding to the motifs recognized by S. aureus sortase A and B, respectively, do not appear to be substrates for SrtBΔN26. SrtBΔN26 also failed to cleave the proposed sorting signal Cell Cycle inhibitor NVQTG from recently characterized collagen binding protein, CbpA. Bars indicate the mean, and error bars represent the standard error (**corresponds to p < 0.01). Analysis of FRET reaction To investigate the importance of the cysteine residue in the proposed

active site of C. difficile SrtB, site-directed mutagenesis was used to replace the cysteine residue at position 209 with an alanine. When the resulting mutant protein SrtBΔN26,C209A was incubated with the FRET peptides, the fluorescent signal fell below the limits of detection (Figure 5), indicating that the cysteine residue at position 209 was essential for the activity of the C. difficile SrtB. Cleavage in the FRET-based assay was also inhibited by the addition of MTSET (Figure 5), a known cysteine protease inhibitor and inhibitor

of sortase function in S. aureus [36,37] and B. anthracis [15]. Figure 5 SrtB ΔN26 activity find more requires a cysteine residue at position 209. To determine if SrtBΔN26 activity depended on the cysteine residue at position 209, a C209A substitution was made to create SrtBΔN26,C209A. This enzyme was inactive against the FRET peptides when compared with SrtBΔN26. Addition at 5 mM of the cysteine protease inhibitor, MTSET, to the reaction also eliminates activity (**corresponds to p < 0.01). The cleavage of the SPKTG, PPKTG, and SPQTG motifs was enhanced at least two-fold by the addition of the two native amino acids immediately downstream of this sequon (data not shown). Analysis of the FRET reaction with these modified peptides revealed Quisqualic acid that SrtBΔN26, cleaves these peptides between the T and G residues. MALDI analysis of d-PVPPKTGDS-e peptide incubated with SrtBΔN26 results in a peptide with a mass of 889 Da, corresponding to the fragment d-PVPPKT-OH (Figure 6, top). The peptide control, incubated without SrtBΔN26, lacked this fragment (Figure 6, bottom). Cleavage between the T and G residues for the d-SDSPKTGDN-e and d-IHSPQTGDV-e peptides was also confirmed (data not shown), indicating that C. difficile SrtB cleaves the (S/P)PXTG motif between the same residues as other functional sortases [4,15,38,39].