Engineering the Substrate Specificity of Staphylococcus aureus Sortase A

The Staphylococcus aureus transpeptidase Sortase A (SrtA) anchors virulence and colonization-associated surface proteins to the cell wall. SrtA selectively recognizes a C-terminal LPXTG motif, whereas the related transpeptidase Sortase B (SrtB) recognizes a C-terminal NPQTN motif. In both enzymes, cleavage occurs after the conserved threonine, followed by amide bond formation between threonine and the pentaglycine cross-bridge of cell wall peptidoglycan. Genetic and biochemical studies strongly suggest that SrtA and SrtB exhibit exquisite specificity for their recognition motifs. To better understand the origins of substrate specificity within these two isoforms, we used sequence and structural analysis to predict residues and domains likely to be involved in conferring substrate specificity. Mutational analyses and domain swapping experiments were conducted to test their function in substrate recognition and specificity. Marked changes in the specificity profile of SrtA were obtained by replacing the β6/β7 loop in SrtA with the corresponding domain from SrtB. The chimeric β6/β7 loop swap enzyme (SrtLS) conferred the ability to acylate NPQTN-containing substrates, with a kcat/Kmapp of 0.0062 ± 0.003 m-1 s-1. This enzyme was unable to perform the transpeptidation stage of the reaction, suggesting that additional domains are required for transpeptidation to occur. The overall catalytic specificity profile (kcat/Kmapp(NPQTN)/kcat/Kmapp(LPETG)) of SrtLS was altered 700,000-fold from SrtA. These results indicate that the β6/β7 loop is an important site for substrate recognition in sortases.