Mutational Analysis of Active Site Residues in the Staphylococcus aureus Transpeptidase SrtA

Abstract

In Staphylococcus aureus, virulence and colonization-associated surface proteins are covalently anchored to the cell wall by the transpeptidase Sortase A (SrtA). In order to better understand the contribution of specific active site residues to substrate recognition and catalysis, we performed mutational analysis of several key residues in the SrtA active site. Analysis of protein stability, kinetic parameters, solvent isotope effects, and pH-rate profiles for key SrtA variants are consistent with a reverse protonated Cys184-His120 catalytic dyad, and implicate a role for Arg197 in formation of an oxyanion hole to stabilize the transition state. In contrast, mutation of Asp185 and Asp186 produced negligible effects on catalysis, and no evidence was found to support the existence of a functional catalytic triad. Mutation of Thr180, Leu181, and Ile182 to alanine produced modest decreases in SrtA activity and led to substrate inhibition. Thermodynamic stability measurements by SUPREX (stability of unpurified proteins from rates of H/D exchange) revealed decreases in conformational stability that correlate with the observed substrate inhibition for each variant, signifying a potential role for the conserved 180TLITC184 motif in defining the active-site architecture of SrtA. In contrast, mutation of Thr183 to alanine led to a significant 1200-fold decrease in kcat, which appears to be unrelated to conformational stability. Potential explanations for these results are discussed, and a revised model for SrtA catalysis is presented.

Crystal Structure of Streptococcus pyogenes Sortase A

“…Transpeptidation Activity-Sp-SrtA ⌬81 activity was determined using an HPLC-based assay previously developed for Sa-SrtA (14,21). For initial activity determination, Sp-SrtA ⌬81 was incubated with 1.0 mM Abz-LPETGG-Dap(Dnp)-NH 2 and 2.0 mM NH 2 -Ala 2 -OH in assay buffer (300 mM Tris, 150 mM NaCl, 5 mM CaCl 2 , pH 7.5).…”

“…The His may act as a general acid to protonate the tetrahedral intermediate, thus facilitating collapse of the transition state and formation of an acyl-enzyme intermediate. Meanwhile, the Arg is thought to either stabilize the short-lived oxyanion-transition state (14) or to be involved in substrate recognition via a hydrogen bond (15). This reverse-protonation mechanism of catalysis, involving a Cys thiolate nucleophile and a His imidazolium general acid (but not a pre-organized ion pair), remains somewhat controversial as the pK a values of the Cys and His in Staphylococcus aureus sortase A (Sa-SrtA, 2 the most extensively studied sortase enzyme) have been measured as ϳ9.4 and 6.2-7.0 (16,17), respectively.…”

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

“…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).…”

The binding mechanism, multiple binding modes, and allosteric regulation of Staphylococcus aureus Sortase A probed by molecular dynamics simulations

“…20 The active sites of all sortases contain a conserved catalytic triad that consists of residues H120, C184, and R197, mutations to each of which have been shown to severely reduce SrtA's catalytic activity. 9,[22][23][24][25] C184 has been established as the activesite nucleophile that covalently attaches to the carbonyl carbon of the SS threonine residue through a ping-pong mechanism. 17,18 R197 appears to stabilize the binding of an SS or an oxyanion intermediate to SrtA, 20,21,24,26 although it has also been suggested to play a role in the deprotonation of C184 or lipid II.…”