The high-affinity peptidoglycan binding domain of Pseudomonas phage endolysin KZ144
The high-affinity peptidoglycan binding domain of Pseudomonas phage endolysin KZ144
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DOI：
10.1016/j.bbrc.2009.03.161
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发表时间：
2009-05-29
影响因子：
3.1
通讯作者：
Lavigne, Rob
中科院分区：
文献类型：
--
作者：
Briers, Yves;Schmelcher, Mathias;Lavigne, Rob
利用内溶素KZ 144（PBDKZ）与绿色荧光蛋白（GFP）的融合蛋白（PBDKZ-GFP），研究了来源于铜绿假单胞菌噬菌体KZ的N-末端肽聚糖结合结构域（PBDKZ）的结合亲和力。结合PBDKZ-GFP分子的光漂白分析后的荧光恢复显示，在15分钟内漂白区域的荧光恢复率低于10%。表面等离子体共振分析证实了这种表观的高结合亲和力，揭示了PBDKZ-肽聚糖相互作用的平衡亲和力常数为2.95 × 10（7）M-1。该独特结构域结合所有测试的革兰氏阴性物种的肽聚糖，用于改善肽聚糖水解酶结构域KMV 36 C的比活性。嵌合肽聚糖水解酶（PBDKZ-KMV 36 C）比天然催化结构域（KMV 36 C）的活性高3倍。这些结果表明，功能结构域的模块化组装是一种合理的方法，以提高从细菌感染革兰氏阴性菌的内溶素的比活性。(C)2009 Elsevier Inc. All rights reserved.
The binding affinity of the N-terminal peptidoglycan binding domain of endolysin KZ144 (PBDKZ), originating from Pseudomonas aeruginosa bacteriophage KZ, has been examined using a fusion protein of PBDKZ and green fluorescent protein (PBDKZ-GFP). A fluorescence recovery after photobleaching analysis of bound PBDKZ-GFP molecules showed less than 10% fluorescence recovery in the bleached area within 15 min. Surface plasmon resonance analysis confirmed this apparent high binding affinity revealing an equilibrium affinity constant of 2.95 x 10(7) M-1 for the PBDKZ-peptidoglycan interaction. This unique domain, which binds to the peptidoglycan of all tested Gram-negative species, was harnessed to improve the specific activity of the peptidoglycan hydrolase domain KMV36C. The chimeric peptidoglycan hydrolase (PBDKZ-KMV36C) exhibits a threefold higher specific activity than the native catalytic domain (KMV36C). These results demonstrate that the modular assembly of functional domains is a rational approach to improve the specific activity of endolysins from phages infecting Gram-negatives. (C) 2009 Elsevier Inc. All rights reserved.