Suvash Chandra OjhaChompounoot ImtongKanungsuk MeetumSomsri SakdeeGerd KatzenmeierChanan AngsuthanasombatMahidol UniversityPrince of Songkla UniversityBiophysics Institute for Research and Development (BIRD)2019-08-232019-08-232018-11-01Protein Expression and Purification. Vol.151, (2018), 106-112104659282-s2.0-85049336438https://repository.li.mahidol.ac.th/handle/20.500.14594/45006© 2018 Elsevier Inc. Lysostaphin, a bacteriolytic toxin from Staphylococcus simulans, is a Zn 2+ -dependent endopeptidase that cleaves pentaglycine cross-bridges found in peptidoglycan of certain Staphylococci. Here, we have investigated a critical influence of Zn 2+ 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 2+ 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 2+ , a marked inhibition in staphylolytic activity was observed. When the pre-mixture was exposed to 1,10-phenanthroline (PNT, a Zn 2+ -chelator), the adverse effect of the exogenous Zn 2+ 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 2+ 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 2+ -binding site found in close proximity to the Zn 2+ -coordinating active site. Overall our results provide more insights into an adverse influence of exogenous Zn 2+ ions on staphylolytic activity of the purified Zn 2+ -dependent endopeptidase lysostaphin, implicating the presence of an extraneous inhibitory metal-binding site.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyArticleSCOPUS10.1016/j.pep.2018.06.013