Journal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Volume 383, Issue 2, Pages 187-191Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2009.03.161
Keywords
Endolysin; Peptidoglycan binding domain; Affinity; Module shuffling; Chimera
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Funding
- Flemish FWO [G.0308.05, 1.5.184.05 N]
- research council of the K.U. Leuven [OT/05147]
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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.
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