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。

Bacteriophage Pub Date : 2013-07-01 Epub Date: 2013-10-02 DOI:10.4161/bact.26564
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
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引用次数: 14

摘要

P128是一种嵌合抗葡萄球菌蛋白,其催化结构域来自葡萄球菌噬菌体K尾相关结构蛋白,细胞壁靶向结构域来自葡萄球菌溶葡萄素。在这项研究中,我们揭示了P128的其他特性,并将其与溶葡萄球菌蛋白进行了比较。溶葡萄球菌素在高温下失活,对其亲本菌株拟南葡萄球菌无活性,而P128具有耐热性,对拟南葡萄球菌有裂解作用,两者作用机制不同。P128催化结构域的选择性突变研究表明,精氨酸和半胱氨酸分别位于第40位和第76位,对P128的葡萄球菌降解活性至关重要,尽管这些氨基酸不是保守残基。与天然P128相比,只有R40S突变体(P301)在酶谱凝胶上具有催化活性,并且具有相似的二级结构,通过圆二色性分析和硅模型评估具有相似的细胞结合特性。P128分子第40位精氨酸残基的突变使Vmax(∆OD600 [mg/min])值显著降低(约270倍),重组溶葡萄球菌蛋白的Vmax值也比未修饰的P128蛋白低(约1.5倍)。P128蛋白的表观Km (KmAPP)和表观KcatAPP (KcatAPP)等动力学参数也与P301和溶葡萄蛋白存在显著差异。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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.

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