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

Abstract

The Staphylococcus aureus transpeptidase sortase A (SrtA) is responsible for anchoring a range of virulence-and colonization-associated proteins to the cell wall. SrtA recognizes substrates that contain a C-terminal LPXTG motif. This sequence is cleaved following the threonine, and an amide bond is formed between the threonine and the pentaglycine cross-bridge of branched lipid II. Previous studies have implicated the ␤6/␤7 loop region of SrtA in LPXTG recognition but have not systematically characterized this domain. To better understand the individual roles of the residues within this loop, we performed alanine-scanning mutagenesis. Val-168 and Leu-169 were found to be important for substrate recognition, and Glu-171 was also found to be important, consistent with its hypothesized role as a Ca 2؉ -binding residue. Gly-167 and Asp-170 were dispensable for catalysis, as was Gln-172. The role of Arg-197 in SrtA has been the subject of much debate. To explore its role in catalysis, we used native chemical ligation to generate semi-synthetic SrtA in which we replaced Arg-197 with citrulline, a non-ionizable analog. This change resulted in a decrease of <3-fold in k cat /K m , indicating that Arg-197 utilizes a hydrogen bond, rather than an electrostatic interaction. Our results are consistent with a model for LPXTG recognition wherein the Leu-Pro sequence is recognized primarily by hydrophobic contacts with SrtA Val-168 and Leu-169, as well as a hydrogen bond from Arg-197. This model contradicts the previously proposed mechanism of binding predicted by the x-ray crystal structure of SrtA.The increasing prevalence of multidrug-resistant Gram-positive bacterial infections has been a cause for great concern (1, 2). Most alarming has been the recent characterization of strains of Staphylococcus aureus which exhibit either intermediate or full resistance to vancomycin, currently the antibiotic of last resort for methicillin-resistant S. aureus infections (3-5). During pathogenesis, Gram-positive bacteria use virulence factors to aid in adherence to host endothelial tissues, evasion of the immune system, and the acquisition of iron from the local environment (6, 7). There has been a great deal of recent interest in the idea of targeting these virulence factors as a possible strategy for the development of new therapeutics for the treatment of 9).Sortases are cysteine transpeptidase enzymes found in a wide-range of Gram-positive pathogens. They catalyze the formation of a covalent linkage between surface proteins containing an LPXTG motif and the peptidoglycan layer of the cell wall (6). Many of these cell wall-anchored proteins function as virulence factors (6, 10). As such, several previous studies have implicated a role for sortases in promoting virulence in several strains of Gram-positive bacteria. Sortase knock-out strains have been shown to have reduced pathogenicity in S. aureus (11), Listeria monocytogenes (12, 13), and in several members of the Streptococcus lineage: S. gordonii (14), S. pneumonia (15), S. pyoge...

A general strategy for the evolution of bond-forming enzymes using yeast display

“…To gain insight into how these mutations improve catalysis, we expressed and purified each sortase single mutant, clones 4.2 and 4.3, and the tetramutant from Escherichia coli, and we measured the saturation kinetics of WT srtA and the mutants using an established HPLC assay (28). The observed kinetic parameters for the WT enzyme closely match those previously reported (26,28). Each single mutation in isolation contributed a small beneficial effect on turnover (k cat ) and more significant beneficial effects on LPETG substrate recognition, lowering the K m LPETG up to threefold ( Table 1).…”

“…None of these four mutations have been reported in previous mutational studies studying the sortase active site and the molecular basis of LPETG substrate recognition (26,27). To gain insight into how these mutations improve catalysis, we expressed and purified each sortase single mutant, clones 4.2 and 4.3, and the tetramutant from Escherichia coli, and we measured the saturation kinetics of WT srtA and the mutants using an established HPLC assay (28).…”

Efficient N‐Terminal Labeling of Proteins by Use of Sortase

“…The efficiency of any given substrate is therefore controlled by the rate of its turnover relative to that for the product. The Michaelis constants for peptide acyl donors are typically in the same range as the substrate concentrations used in this study; [10,15] the relative rate of reaction is therefore determined by the specificity constant, k cat /K m . We suggest that this factor accounts for the success of the depsipeptides under the conditions used in this study.…”

The Structure of the Staphylococcus aureus Sortase-Substrate Complex Reveals How the Universally Conserved LPXTG Sorting Signal Is Recognized

“…The lone exception is the crystal structure of SrtA ⌬N59 bound to an LPETG peptide (31). However, in this structure the peptide substrate is bound nonspecifically (see below) (32,39).…”

“…These residues play a critical role in catalysis, since their mutation in SrtA causes severe reductions in enzyme activity (16, 26 -30 (28), deprotonating lipid II (31), or stabilizing the binding of either the LPXTG sorting signal (28,32) or oxyanion intermediates (31,32). Different functions have also been proposed for His 120 .…”