Purification and characterization of the antibacterial peptidase lysostaphin from Staphylococcus simulans: Adverse influence of Zn on bacteriolytic activity.

Lysostaphin, a bacteriolytic toxin from Staphylococcus simulans, is a Zn-dependent endopeptidase that cleaves pentaglycine cross-bridges found in peptidoglycan of certain Staphylococci. Here, we have investigated a critical influence of Zn ions on lysostaphin-induced bioactivity. Initially, we succeeded in producing a large amount with high purity of the 28-kDa His-tagged mature lysostaphin via soluble expression in Escherichia coli and subsequent purification via immobilized-Ni affinity chromatography (IMAC). The purified monomeric bacteriocin exhibited concentration-dependent bioactivity against S. aureus and its methicillin-resistant strain through cell-wall hydrolysis rather than membrane perturbation. Following pre-incubation of the purified lysostaphin with exogenous Zn, a marked inhibition in staphylolytic activity was observed. When the pre-mixture was exposed to 1,10-phenanthroline (PNT, a Zn-chelator), the adverse effect of the exogenous Zn on bioactivity was greatly decreased. Conversely, lysostaphin pre-treated with excess PNT retained relatively high bioactivity, indicating ineffective chelation of PNT to detach the catalytic Zn from the active-site pocket. Structural analysis of the lysostaphin-catalytic domain together with amino acid sequence alignments of lysostaphin-like endopeptidases revealed a potential extraneous Zn-binding site found in close proximity to the Zn-coordinating active site. Overall our results provide more insights into an adverse influence of exogenous Zn ions on staphylolytic activity of the purified Zn-dependent endopeptidase lysostaphin, implicating the presence of an extraneous inhibitory metal-binding site.