The different classes of sortase share a common ping-pong bi-bi transpeptidation reaction mechanism, according to which the sortase first binds the 5-amino acid recognition motif located in the C-terminal region of the substrate protein (Frankel et al., 2005, 2007).The sortases then form thioacyl?enzyme intermediates between the catalytic cysteine and the substrate threonine, which are resolved by a nucleophilic attack by components of the bacterial cell wall. Since, after the discovery of different classes of sortases, Staphylococcus aureus sortase A (SrtA) has been the prototype for understanding the mechanism of action of these enzymes (Mazmanian et al., 1999). In Staphylococcus aureus, sortase A (SrtA) recognizes the pentaglycine sequence on the surface proteins, which are being secreted through the cytoplasmic membrane. The pentaglycine sequence contains an LPXTG motif at the C-terminus of the protein. The SrtA cleaves the scissile bond between threonine and glycine residues to form an acyl-enzyme intermediate which subsequently transfers the carboxyl of threonine which is amide-linked to the pentaglycine cross-bridge of lipid II (Marraffini et al.
, 2006). Finally, the lipid II- surface protein complex gets incorporated into the peptidoglycan by means of transglycosylation and transpeptidation reactions (Paterson and Mitchell, 2004; Spirig et al., 2011). The sortase enzyme accepts the nucleophiles which might vary in different Gram-positive bacteria as the composition of peptidoglycan layers in the cell envelope vary from strain to strain. For example, Diaminopimelic acid which cross-bridges the peptidoglycan in Bacillus anthracis is thought to be the point of attachment for the sortase substrate proteins in Bacillus strains.14–16 In absence of a dedicated nucleophile, the acyl-enzyme intermediate complex was shown to be hydrolyzed, resulting in cleavage of the recognition sequence without formation of a new peptide bond.12