Sortase

Sortases are a family of cysteine transpeptidases found in most Gram-positive bacteria. They catalyze covalent anchoring of surface proteins to the cell wall by recognizing a conserved LPXTG motif in their substrates, with sortase A (SrtA) being the best studied member. Through this activity, sortases enable the cell surface display of adhesins, enzymes, and pilus-associated proteins.

Mechanism aside from anchoring: The enzyme cleaves the LPXTG motif between the threonine and glycine, forming a thioester intermediate with its active-site cysteine. A free amino group—typically from the peptidoglycan cross-bridge or an acceptor protein—attacks the intermediate to form a stable amide bond, covalently attaching the substrate to the cell wall.

Substrate specificity and diversity: Different sortases recognize distinct sorting signals. SrtA attaches a broad range of LPXTG-containing proteins to the cell wall. Other sortases, such as SrtC, participate in pilus assembly by polymerizing pilin subunits and linking them to the wall. Some bacteria encode multiple sortases with specialized roles, reflecting the diversity of surface proteins.

Structure, localization, and regulation: Sortases are anchored to the cytoplasmic membrane, with the catalytic domain accessible for substrate processing. The active site typically involves a cysteine, histidine, and aspartate residues forming a catalytic motif. Calcium ions often enhance the activity of several sortases.

Applications and relevance: Sortase-mediated ligation, or sortagging, exploits the LPXTG recognition to attach glycine-terminated probes or proteins to substrates bearing N-terminal glycines, enabling programmable protein conjugation, peptide cyclization, and surface labeling. Clinically, sortases contribute to virulence in some pathogens, making them potential targets for antimicrobial strategies.