{"title":"噬菌体衍生的嵌合重组葡萄球菌溶解蛋白P128的性质和突变研究:与重组溶葡萄球菌蛋白的比较。","authors":"Sanjeev Rajagopalan Saravanan, Vivek Daniel Paul, Shilpa George, Sudarson Sundarrajan, Nirmal Kumar, Madhavi Hebbur, Naveen Kumar, Ananda Veena, Uma Maheshwari, Chemira Biddappa Appaiah, Muralidharan Chidambaran, Anuradha Gopal Bhat, Sukumar Hariharan, Sriram Padmanabhan","doi":"10.4161/bact.26564","DOIUrl":null,"url":null,"abstract":"<p><p>P128 is a chimeric anti-staphylococcal protein having a catalytic domain from a <i>Staphylococcus</i> bacteriophage K tail associated structural protein and a cell wall targeting domain from the <i>Staphylococcus</i> bacteriocin-lysostaphin. In this study, we disclose additional properties of P128 and compared the same with lysostaphin. While lysostaphin was found to get inactivated by heat and was inactive on its parent strain <i>S. simulans</i> biovar <i>staphylolyticus</i>, P128 was thermostable and was lytic towards <i>S. simulans</i> biovar <i>staphylolyticus</i> demonstrating a difference in their mechanism of action. Selected mutation studies of the catalytic domain of P128 showed that arginine and cysteine, at 40th and 76th positions respectively, are critical for the staphylolytic activity of P128, although these amino acids are not conserved residues. In comparison to native P128, only the R40S mutant (P301) was catalytically active on zymogram gel and had a similar secondary structure, as assessed by circular dichroism analysis and in silico modeling with similar cell binding properties. Mutation of the arginine residue at 40th position of the P128 molecule caused dramatic reduction in the V<sub>max</sub> (∆OD<sub>600</sub> [mg/min]) value (nearly 270 fold) and the recombinant lysostaphin also showed lesser V<sub>max</sub> value (nearly 1.5 fold) in comparison to the unmodified P128 protein. The kinetic parameters such as apparent K<sub>m</sub> (K<sub>m</sub><sup>APP</sup>) and apparent K<sub>cat</sub> (K<sub>cat</sub><sup>APP</sup>) of the native P128 protein also showed significant differences in comparison to the values observed for P301 and lysostaphin.</p>","PeriodicalId":8686,"journal":{"name":"Bacteriophage","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2013-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.4161/bact.26564","citationCount":"14","resultStr":"{\"title\":\"Properties and mutation studies of a bacteriophage-derived chimeric recombinant staphylolytic protein P128: Comparison to recombinant lysostaphin.\",\"authors\":\"Sanjeev Rajagopalan Saravanan, Vivek Daniel Paul, Shilpa George, Sudarson Sundarrajan, Nirmal Kumar, Madhavi Hebbur, Naveen Kumar, Ananda Veena, Uma Maheshwari, Chemira Biddappa Appaiah, Muralidharan Chidambaran, Anuradha Gopal Bhat, Sukumar Hariharan, Sriram Padmanabhan\",\"doi\":\"10.4161/bact.26564\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>P128 is a chimeric anti-staphylococcal protein having a catalytic domain from a <i>Staphylococcus</i> bacteriophage K tail associated structural protein and a cell wall targeting domain from the <i>Staphylococcus</i> bacteriocin-lysostaphin. In this study, we disclose additional properties of P128 and compared the same with lysostaphin. While lysostaphin was found to get inactivated by heat and was inactive on its parent strain <i>S. simulans</i> biovar <i>staphylolyticus</i>, P128 was thermostable and was lytic towards <i>S. simulans</i> biovar <i>staphylolyticus</i> demonstrating a difference in their mechanism of action. Selected mutation studies of the catalytic domain of P128 showed that arginine and cysteine, at 40th and 76th positions respectively, are critical for the staphylolytic activity of P128, although these amino acids are not conserved residues. In comparison to native P128, only the R40S mutant (P301) was catalytically active on zymogram gel and had a similar secondary structure, as assessed by circular dichroism analysis and in silico modeling with similar cell binding properties. Mutation of the arginine residue at 40th position of the P128 molecule caused dramatic reduction in the V<sub>max</sub> (∆OD<sub>600</sub> [mg/min]) value (nearly 270 fold) and the recombinant lysostaphin also showed lesser V<sub>max</sub> value (nearly 1.5 fold) in comparison to the unmodified P128 protein. The kinetic parameters such as apparent K<sub>m</sub> (K<sub>m</sub><sup>APP</sup>) and apparent K<sub>cat</sub> (K<sub>cat</sub><sup>APP</sup>) of the native P128 protein also showed significant differences in comparison to the values observed for P301 and lysostaphin.</p>\",\"PeriodicalId\":8686,\"journal\":{\"name\":\"Bacteriophage\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2013-07-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.4161/bact.26564\",\"citationCount\":\"14\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Bacteriophage\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.4161/bact.26564\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2013/10/2 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Bacteriophage","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.4161/bact.26564","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2013/10/2 0:00:00","PubModel":"Epub","JCR":"","JCRName":"","Score":null,"Total":0}
Properties and mutation studies of a bacteriophage-derived chimeric recombinant staphylolytic protein P128: Comparison to recombinant lysostaphin.
P128 is a chimeric anti-staphylococcal protein having a catalytic domain from a Staphylococcus bacteriophage K tail associated structural protein and a cell wall targeting domain from the Staphylococcus bacteriocin-lysostaphin. In this study, we disclose additional properties of P128 and compared the same with lysostaphin. While lysostaphin was found to get inactivated by heat and was inactive on its parent strain S. simulans biovar staphylolyticus, P128 was thermostable and was lytic towards S. simulans biovar staphylolyticus demonstrating a difference in their mechanism of action. Selected mutation studies of the catalytic domain of P128 showed that arginine and cysteine, at 40th and 76th positions respectively, are critical for the staphylolytic activity of P128, although these amino acids are not conserved residues. In comparison to native P128, only the R40S mutant (P301) was catalytically active on zymogram gel and had a similar secondary structure, as assessed by circular dichroism analysis and in silico modeling with similar cell binding properties. Mutation of the arginine residue at 40th position of the P128 molecule caused dramatic reduction in the Vmax (∆OD600 [mg/min]) value (nearly 270 fold) and the recombinant lysostaphin also showed lesser Vmax value (nearly 1.5 fold) in comparison to the unmodified P128 protein. The kinetic parameters such as apparent Km (KmAPP) and apparent Kcat (KcatAPP) of the native P128 protein also showed significant differences in comparison to the values observed for P301 and lysostaphin.