Chandra Ojha Suvash, Imtong Chompounoot, Meetum Kanungsuk, Sakdee Somsri, Katzenmeier Gerd, Angsuthanasombat Chanan
Bacterial Toxin Research Innovation Cluster (BRIC), Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom, 73170, Thailand.
Division of Biology, Department of Science, Faculty of Science and Technology, Prince of Songkla University, Pattani, 94000, Thailand.
Protein Expr Purif. 2018 Nov;151:106-112. doi: 10.1016/j.pep.2018.06.013. Epub 2018 Jun 23.
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.
溶葡萄球菌素是一种来自模仿葡萄球菌的溶菌毒素，是一种锌依赖性内肽酶，可切割某些葡萄球菌肽聚糖中发现的五甘氨酸交联桥。在此，我们研究了锌离子对溶葡萄球菌素诱导的生物活性的关键影响。最初，我们通过在大肠杆菌中可溶性表达，随后通过固定化镍亲和色谱法（IMAC）纯化，成功大量生产了高纯度的28 kDa组氨酸标签成熟溶葡萄球菌素。纯化的单体细菌素通过细胞壁水解而非膜扰动，对金黄色葡萄球菌及其耐甲氧西林菌株表现出浓度依赖性生物活性。将纯化的溶葡萄球菌素与外源锌预孵育后，观察到葡萄球菌溶解活性明显受到抑制。当预混合物暴露于1,10-菲咯啉（PNT，一种锌螯合剂）时，外源锌对生物活性的不利影响大大降低。相反，用过量PNT预处理的溶葡萄球菌素保留了相对较高的生物活性，表明PNT螯合无效，无法从活性位点口袋中分离催化锌。对溶葡萄球菌素催化结构域的结构分析以及溶葡萄球菌素样内肽酶的氨基酸序列比对揭示了在与锌配位活性位点紧邻处发现的一个潜在的额外锌结合位点。总体而言，我们的结果为外源锌离子对纯化的锌依赖性内肽酶溶葡萄球菌素的葡萄球菌溶解活性的不利影响提供了更多见解，暗示存在一个额外的抑制性金属结合位点。
