Ke ChenSuvash Chandra OjhaChompounoot ImtongAung Khine LinnHui Chun LiCharoensri ThonabulsombatChanan AngsuthanasombatTzu Chi UniversityMahidol UniversityInstitute of Molecular Biosciences, Mahidol UniversityPrince of Songkla UniversityBiophysics Institute for Research and Development (BIRD)Hospital of Southwest Medical University2022-08-042022-08-042021-01-01Protein and Peptide Letters. Vol.28, No.2 (2021), 20-2818755305092986652-s2.0-85103744719https://repository.li.mahidol.ac.th/handle/20.500.14594/76389Background: 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 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 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.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyArticleSCOPUS10.2174/0929866527666200613221359