Identification of sortase substrates by specificity profiling

Abstract

Sortases catalyze the attachment of surface proteins to the peptidoglycan layer of gram-positive bacteria and further represent powerful tools of protein chemistry. During catalysis sortases cleave a donor substrate containing the LPxTG (x=any amino acid) sorting motif under formation of an enzyme-bound thioester and ligate this intermediate to an acceptor protein containing an N-terminal glycine residue. In addition to the well-established sortase A of Staphylococcus aureus several homologs of this enzyme have been identified in the genomes of gram-positive bacteria. We have profiled the specificity of seven sortases of Staphylococci and Streptococci origin and observed that sortases of the latter class displayed a more relaxed specificity for donor and acceptor substrates than their Staphylococci counterparts. Streptococci sortases prefer an LPKLG donor substrate sequence compared to the canonical sorting motif LPKTG. These findings might facilitate the use of Streptococci sortases as tools of protein chemistry.

Keywords: Protein labeling; Sortase; Sortase-mediated ligation; Staphylococcus aureus; Streptococcus pyogenes.