Sortases are Gram-positive bacterial extracellular cysteine transpeptidases responsible for covalently attaching secreted proteins to the peptidoglycan cell wall. The name is derived from the role the enzymes play in the protein sorting pathway, ‘sorting’ cell surface proteins into the cell wall compartment of Gram-positive bacteria. This Sortase A-mediated ligation is known as sortase-mediated ligation (SML) or sortagging. Sortase A (SrtA) enzymes are known as the ‘housekeeping’ sortases as they are ubiquitous in almost all Gram-positive bacteria. Sortase B (SrtB) enzymes are responsible for sorting a single substrate containing the consensus sequence NPQTN. The structure of the SrtA and SrtB protein core is an eight-stranded beta-barrel that has five alpha-helices. Sortase C (SrtC) enzymes are responsible for polymerizing pilin proteins to form pili. Sortase D (SrtD) enzymes are responsible for sorting proteins involved in sporulation.

Sortase A (SrtA) recognise surface proteins with the sequence motif LPXTG, where X is any amino acid, in the C-terminus and subsequently cleave it after the threonine. A covalent intermediate is formed between the threonine and the active site cysteine.  A transpeptidation reaction then catalyses the formation of an amide bond between the new C-terminal threonine and the N-terminal amino group of oligo-glycines in peptidoglycan or pseudopeptidoglycan to covalently anchor them of the bacterial cell wall. SrtA contain three conserved residues within their active site: His120, Cys184, and Arg197. Cys184 forms a covalent linkage to the sorting signal. His120 can function to activate Cys184 by forming an imidazolium-thiolate ion-pair or act as a general base instead of the ion-pair. The Arg197 is crucial for stabilising the binding of the substrate in the active site by donating hydrogen bonds from its guanidine group to the LP and TG carbonyls of the LPXTG substrate motif, depending on the protonation state of the catalytic Cys184-His120 dyad. Substrate binding leads to conformational change in the active site. The structures of the enzyme–substrate complexes vary depending on the sorting signal.

 The transpeptidase activity of SrtA can be applied to catalyse in vivo and in vitro ligation of proteins or peptide substrates containing a C-terminal LPETG motif with fragments containing several N-terminal glycines. The versatile and broad application of SrtA mediated ligation can be used for protein modification, synthesis of protein–polymer conjugates and immobilization of proteins on surfaces.