ABSTRACT
Sortase-catalyzed transacylation reactions are widely used for the construction of non-natural protein derivatives. However, the most commonly used enzyme for these strategies (sortase A from Staphylococcus aureus) is limited by its narrow substrate scope. To expand the range of substrates compatible with sortase-mediated reactions, we characterized the in vitro substrate preferences of eight sortaseâ A homologues. From these studies, we identified sortaseâ A enzymes that recognize multiple substrates that are unreactive toward sortaseâ A from S.â aureus. We further exploited the ability of sortaseâ A from Streptococcus pneumoniae to recognize an LPATS substrate to perform a site-specific modification of the N-terminal serine residue in the naturally occurring antimicrobial peptide DCD-1L. Finally, we unexpectedly observed that certain substrates (LPATXG, X=Nle, Leu, Phe, Tyr) were susceptible to transacylation at alternative sites within the substrate motif, and sortase A from S.â pneumoniae was capable of forming oligomers. Overall, this work provides a foundation for the further development of sortase enzymes for use in protein modification.