Catalytic Mechanism and Roles of Arg197 and Thr183 in the Staphylococcus aureus Sortase A Enzyme

Abstract

The sortase A enzyme, which catalyzes the peptidoglycan cell wall anchoring reaction of LPXTG surface proteins, has been proposed to be a universal target for therapeutic agents against Gram-positive bacteria. The catalytic mechanism of the Staphylococcus aureus sortase A enzyme has been systematically studied using molecular dynamics simulations, ONIOM(DFT:MM) calculations, and QM/MM charge deletion analysis. The catalytic roles of Arg197 and Thr183 were analyzed. Our calculations show that Arg197 has several important roles in the mechanism. It is crucial for substrate binding, and is capable of reversible shift of its hydrogen bonds between the LP and TG carbonyls of the LPXTG substrate motif, depending on the protonation state of the catalytic Cys184-His120 dyad. Arg197 stabilizes the catalytic dyad in the active ion pair form but at the same time raises the barrier to acylation by approximately 8 kcal/mol. Thr183 is also essential for the catalytic reaction in that it correspondingly lowers the barrier by the same amount via electrostatic interactions. The catalytic mechanism proceeds via proton transfer from His120, followed by nucleophilic attack from the thiolate anion of Cys184. The data thus supports the proposed reverse protonation mechanism, and disproves the hypothesis of the Arg197 generating an oxyanion hole to stabilize the tetrahedral intermediate of the reaction.

Structural and Computational Studies of the Staphylococcus aureus Sortase B-Substrate Complex Reveal a Substrate-stabilized Oxyanion Hole

“…Conformations of the SrtA-LPAT complex in which the threonine side chain of the sorting signal forms interactions with the active site arginine were obtained without major rearrangement of the structure of the enzyme. Interestingly, a previous MD study based on the SrtA-LPAT* NMR structure reports that the active site arginine residue functions only to position the sorting signal substrate by hydrogen bonding to its backbone carbonyl atoms at positions P2 and P4 (25). Our work is compatible with this conclusion but suggests that this stabilizing interaction will only occur when the P1 Thr samples the out position that is presumably not catalytically active.…”

Sortase Transpeptidases: Structural Biology and Catalytic Mechanism

“…Comparative simulations with LPAT, APAT, and LAAT substrates demonstrated that contacts between the leucine side chain and SaSrtA contribute to stabilize the β 6/ β 7 loop, whereas the kink that is induced by the proline appears to be essential for recognition. In another study, Tian and Eriksson performed simulations in which His120 and Cys184 were in their zwitterionic and neutral forms (Tian & Eriksson, 2011). Their results showed that Arg197 adopts distinct conformations based upon the charged state of the protein, which helps to stabilize the catalytically active form.…”

“…This process is presumably assisted by the conserved active site histidine residue, which may act as a general acid to protonate the amino leaving group. Beyond stabilizing the oxyanion intermediate, the side chain of the active site arginine residue may orient the substrate in the active site by forming a hydrogen bond to the backbone carbonyl atom in the P2 residue (Chan et al, 2015; Suree et al, 2009; Tian & Eriksson, 2011). In the next step of the reaction, a secondary substrate bearing an amine group enters the active site and is presumably deprotonated by the active site histidine residue to facilitate its nucleophilic attack on the thioacyl bond (Fig.…”

Sortase A: An ideal target for anti-virulence drug development

“…The authors investigated the interactions between the diarylacrylonitrile derivative and the SrtA enzyme (PDB:1IJA) using molecular modelling. Although DMMA does not form an interaction with Cys 184, which is crucial for transpeptidation activity, the nitrile group forms a hydrogen bond with the catalytic residue Arg 197 [40], and the phenyl rings form strong lipophilic interactions with amino acids in the large hydrophobic binding pocket of the SrtA active site. A subsequent study examined the inhibitory effects of DMMA in vivo providing the first data of SrtA inhibitor use in animals [41].…”