Engineering the Substrate Specificity of Staphylococcus aureus Sortase A

Abstract

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 ␤6/␤7 loop in SrtA with the corresponding domain from SrtB. The chimeric ␤6/␤7 loop swap enzyme (SrtLS) conferred the ability to acylate NPQTNcontaining substrates, with a k cat /K m app 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 (k cat /K m app (NPQTN)/k cat /K m app (LPETG)) of SrtLS was altered 700,000-fold from SrtA. These results indicate that the ␤6/␤7 loop is an important site for substrate recognition in sortases.

Crystal Structure of Streptococcus pyogenes Sortase A

“…The ␤ 6 /␤ 7 loop of sortases is important in determining substrate selectivity, and therefore, this region is important for enzyme activity (10,15). In the structures of Sa-SrtA ⌬59 this loop is disordered (high B-factors or not modeled in crystal structures (12) or mobile in NMR structures (18,24,25)).…”

“…The sorting signal that targets proteins for cell surface attachment is located at the C terminus of substrates and comprises a pentapeptide motif, typically LPXTG (where X is any amino acid), followed by a hydrophobic region and a tail of positively charged residues that locates the substrate to the cell surfacefollowingsecretion (2,9).Inonecurrentmodelofsortasedependent transpeptidation, the LPXTG motif is specifically recognized by the enzyme (10), and the thiolate group of an essential active site Cys attacks the scissile Thr-Gly bond (1,11). Two additional absolutely conserved residues are located in the active site, a His and an Arg (12,13).…”

Mutagenesis Studies of Substrate Recognition and Catalysis in the Sortase A Transpeptidase from Staphylococcus aureus

“…Although the importance of the ␤6/␤7 loop in LPXTG recognition has been established (29,30), the exact mechanism by which it does so has not been shown, nor have the contributions of individual residues within the ␤6/␤7 loop been investigated in a systematic way. Additionally, the role of Arg-197 in catalysis by SrtA has remained somewhat a mystery.…”

“…The SrtA substrate peptide Abz-LPETGG-Dap(DNP)-NH 2 was synthesized using the Fmoc/piperidine strategy on 5-(4-Fmoc-aminomethyl-3,5-dimethoxyphenoxy)valeric acidmethylbenzhydrylamine resin at 0.25-mmol scale. Peptides were synthesized and purified using previously established methods (27,30). Purified peptides were lyophilized to dryness, and product identities were confirmed by MALDI-TOF mass spectrometry.…”

“…Third, this loop domain differs between SrtA and SrtB, which recognizes an NPQTN sorting signal. It was shown recently that swapping the ␤6/␤7 loop from SrtB into SrtA was sufficient to alter the specificity of SrtA by over 700,000-fold, directly implicating this loop in substrate discrimination (30).…”