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 Zn2+-dependent endopeptidase, but its bacteriolytic activity could be affected
by exogenously added Zn2+.
Objective: To gain greater insights into structural and functional impacts of Zn2+and Ni2+on Lss-induced
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 Zn2+-treated Lss and S. aureus peptidoglycans. Metal ionbinding
prediction and intermolecular docking were used to locate an extraneous Zn2+-binding site.
Results: A drastic decrease in Lss bioactivity against S. aureus and MRSA was revealed only when
treated with Zn2+, but not Ni2+, albeit no negative effect of diethyldithiocarbamate—Zn2+-chelator
on Lss-induced bioactivity. No severe conformational change was observed for Lss incubated with
exogenous Zn2+ or Ni2+. Lss pre-treated with Zn2+ 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 Zn2+, but not Ni2+, preferably interacted with a potential
extraneous Zn2+-binding site (His253, Glu318 and His323) placed near the Zn2+-coordinating Lssactive
site within the catalytic (CAT) domain.
Conclusion: Our present data signify the adverse influence of exogenous Zn2+ ions on Lss-induced
staphylolytic activity through the exclusive presence within the CAT domain of an extraneous inhibitory
Zn2+-binding site, without affecting the CWT activity.