Inhibition and catalytic mechanism of the Staphylococcus aureus sortase transpeptidase, SrtA

Sortase enzymes play a key role in the pathogenesis of Gram-positive bacteria by catalyzing the covalent linkage of surface-protein virulence and colonization factors to the peptidoglycan layer of the bacterial cell wall. For this reason, S. aureus SrtA has emerged as an attractive target for the development of novel anti-infective agents. SrtA catalyzes the cleavage of surface-protein substrates between the threonine and glycine of a conserved C-terminal LPXTG motif and subsequent amide bond formation between threonine and the peptidoglycan precursor Gly5-Lipid II. Although SrtA active-site residues Cys184 and His120 have been demonstrated to be essential for catalysis, repeated efforts to inhibit SrtA, with a cysteine protease-like mechanism in mind, have yet to yield potent and selective SrtA inhibitors. Inhibitor development is further complicated by the proposal of three conflicting mechanistic models for SrtA catalysis. Initially we explored two alternate strategies for inhibiting SrtA-mediated surface protein display. A series of small-molecule vinyl sulfones were found to irreversibly inactivate SrtA by covalently modifying the active-site nucleophile Cys184. Although kinetic analyses revealed that small-molecule vinyl sulfones are inefficient SrtA inhibitors, in vivo studies suggest that they hold promise for derivatization into viable mimetics. In a separate approach, ramoplanin and vancomycin were demonstrated to inhibit S. aureus surface protein processing, a finding descriptive of a novel mechanism of action for these Lipid II-binding antibiotics. Next, to shed light on the existing controversy surrounding the SrtA catalytic mechanism, we performed detailed kinetic analyses of wild-type SrtA coupled with site-directed mutagenesis of active-site residues. Our findings provide convincing support for a reverse protonation mechanism in which the active form of SrtA, with oppositely protonated Cys184 thiolate and His120 imidazolium, represents a minor, yet highly active, percent of the total enzyme population. We also propose a role for Arg197 in transition-state stabilization and begin to describe the importance of the conserved active-site TLITC motif. This clarification of SrtA mechanism is expected to facilitate the future development of potent sortase inhibitors