The high-affinity peptidoglycan binding domain of Pseudomonas phage endolysin KZ144.
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.
Department of Biosystems, Division of Gene Technology, Katholieke Universiteit Leuven, Leuven, Belgium.
Yves
Y
Briers
Mathias
M
Schmelcher
Martin J
MJ
Loessner
Jelle
J
Hendrix
Yves
Y
Engelborghs
Guido
G
Volckaert
Rob
R
Lavigne
D020134:Catalytic Domain; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D010457:Peptidoglycan / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017105:Pseudomonas Phages / Q000201:enzymology*; D011550:Pseudomonas aeruginosa / Q000821:virology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D010457:Peptidoglycan; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010450:Endopeptidases; C001143:endolysin