Molecular Insights into Zn Inhibition of the Antibacterial Endopeptidase Lysostaphin from .

Background: Mature lysostaphin (~28-kDa Lss) from Staphylococcus simulans proves effective in killing methicillin-resistant Staphylococcus aureus (MRSA) which is endemic in hospitals worldwide. Lss is Zn-dependent endopeptidase, but its bacteriolytic activity could be affected by exogenously added Zn.

Objective: To gain greater insights into structural and functional impacts of Znand Nion Lss-induced bioactivity.

Methods: Lss purified via immobilized metal ion-affinity chromatography was assessed for bioactivity using turbidity reduction assays. Conformational change of metal ion-treated Lss was examined by circular dichroism and intrinsic fluorescence spectroscopy. Co-sedimentation assay was performed to study interactions between Zn-treated Lss and S. aureus peptidoglycans. Metal ionbinding prediction and intermolecular docking were used to locate an extraneous Zn-binding site.

Results: A drastic decrease in Lss bioactivity against S. aureus and MRSA was revealed only when treated with Zn, but not Ni, albeit no negative effect of diethyldithiocarbamate-Zn-chelator on Lss-induced bioactivity. No severe conformational change was observed for Lss incubated with exogenous Zn or Ni. Lss pre-treated with Zn efficiently bound to S. aureus cell-wall peptidoglycans, suggesting non-interfering effect of exogenous metal ions on cell-wall targeting (CWT) activity. In silico analysis revealed that exogenous Zn, but not Ni, preferably interacted with a potential extraneous Zn-binding site (His253, Glu318 and His323) placed near the Zn-coordinating Lssactive site within the catalytic (CAT) domain.

Conclusion: Our present data signify the adverse influence of exogenous Zn ions on Lss-induced staphylolytic activity through the exclusive presence within the CAT domain of an extraneous inhibitory Zn-binding site, without affecting the CWT activity.