裂解酶LysGH15的CHAP片段与底物互作的关键位点

发布时间：2018-06-29 23:28

本文选题：分子对接 + 噬菌体裂解酶　；参考：《中国兽医学报》2017年03期

【摘要】：金黄色葡萄球菌噬菌体裂解酶LysGH15对金葡菌表现出广谱高效的抗菌活性,本课题组已深入研究其三维结构,其中CHAP片段是LysGH15起催化作用的关键活性片段。本试验进一步通过分子模拟和对接的方法预测出CHAP与底物多肽DGlnNH2-LLys-DAla-5Gly结合的"口袋"结构,该结构由D33、S35、F36、Y50、R71、T72及N75这些氨基酸位点组成。由于脂肪族非极性氨基酸丙氨酸侧链最短且不带电荷,因此将这些位点均突变为丙氨酸,逐一研究它们对CHAP片段催化活性的影响:圆二色谱试验结果说明突变体二级结构并未发生变化;酶谱试验结果定性的说明F36突变为丙氨酸后突变体蛋白F36A的活性完全丧失,而其他位点的氨基酸突变后蛋白活性并未发生明显改变;细菌浊度测定和菌落计数试验进一步证明突变体F36A活性完全丧失,而其他突变体活性不变;鉴于F36位于"口袋"结构的"口"的部位,36位氨基酸很可能参与到CHAP与底物多肽的结合。本试验鉴定了LysGH15的CHAP与底物多肽互作过程中起关键作用的氨基酸位点。
[Abstract]:The bacteriophage lyase LysGH15 of Staphylococcus aureus showed broad-spectrum and high-efficient antibacterial activity against Staphylococcus aureus. The three-dimensional structure of LysGH15 has been studied in depth, and chap fragment is the key active fragment of LysGH15. In this experiment, the pocket structure of chap combined with DGlnNH2-Lys-DAla-5Gly was predicted by molecular simulation and docking. The structure was composed of amino acid sites such as D33, S35F36, Y50, R71T72 and N75, which were composed of two amino acids, DGlnNH2-Lys-DAla-5Gly, DGlnNH2-Lys-DAla-5Gly. Because the alanine side chain of aliphatic nonpolar amino acid is the shortest and without charge, these sites are mutated to alanine. The results of circular dichroism showed that the secondary structure of the mutant did not change, and the result of zymogram showed that the activity of protein F36A was completely lost after the mutation of F36 into alanine. But the protein activity of other amino acid mutation did not change obviously, the bacterial turbidity test and colony count test further proved that the activity of the mutant F36A was completely lost, while the activity of other mutants was unchanged. Since F 36 is located in the "mouth" of the "pocket" structure, the 36 amino acid is likely to be involved in the binding of chap to the substrate polypeptide. The amino acid sites of LysGH15 were identified which played a key role in the interaction between chap and substrate peptides.
【作者单位】：吉林大学动物医学学院;吉林大学生命科学学院;江苏高校动物重要疫病与人兽共患病防控协同创新中心;
【基金】：国家自然科学基金资助项目(31130072);国家自然科学基金面上资助项目(31572553);国家自然科学基金青年资助项目(31502103)
【分类号】：S852.61

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