Briers Yves, Schmelcher Mathias, Loessner Martin J, Hendrix Jelle, Engelborghs Yves, Volckaert Guido, Lavigne Rob
Department of Biosystems, Division of Gene Technology, Katholieke Universiteit Leuven, Leuven, Belgium.
Biochem Biophys Res Commun. 2009 May 29;383(2):187-91. doi: 10.1016/j.bbrc.2009.03.161. Epub 2009 Apr 5.
The binding affinity of the N-terminal peptidoglycan binding domain of endolysin KZ144 (PBD(KZ)), originating from Pseudomonas aeruginosa bacteriophage varphiKZ, has been examined using a fusion protein of PBD(KZ) and green fluorescent protein (PBD(KZ)-GFP). A fluorescence recovery after photobleaching analysis of bound PBD(KZ)-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 PBD(KZ)-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 (PBD(KZ)-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.
利用源自铜绿假单胞菌噬菌体φKZ的内溶素KZ144的N端肽聚糖结合结构域（PBD(KZ)）与绿色荧光蛋白的融合蛋白（PBD(KZ)-GFP），检测了该结构域的结合亲和力。对结合的PBD(KZ)-GFP分子进行光漂白后荧光恢复分析，结果显示在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明显的高结合亲和力，揭示了PBD(KZ)与肽聚糖相互作用的平衡亲和常数为2.95×10⁷M⁻¹。这个能与所有测试革兰氏阴性菌的肽聚糖结合的独特结构域，被用于提高肽聚糖水解酶结构域KMV36C的比活性。嵌合肽聚糖水解酶（PBD(KZ)-KMV36C）的比活性比天然催化结构域（KMV36C）高3倍。这些结果表明，功能结构域的模块化组装是提高感染革兰氏阴性菌的噬菌体来源内溶素比活性的合理方法。
