JAC Advance Access originally published online on January 28, 2003
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Journal of Antimicrobial Chemotherapy (2003) 51, 531-543
© 2003 The British Society for Antimicrobial Chemotherapy
Identification of novel inhibitors of Pseudomonas aeruginosa MurC enzyme derived from phage-displayed peptide libraries
1 Faculté de Médecine and 2 Département de Biochimie, Centre de Recherche sur la Fonction, Structure et Ingénierie des Protéines, Pavillon Charles-Eugène-Marchand, Faculté des Sciences et Génie, Université Laval, Sainte-Foy, Québec, Canada G1K 7P4; 3 Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada K1A 0R6
Received 14 June 2002; returned 13 August 2002; revised 23 September 2002; accepted 24 September 2002
Objectives: The machinery of peptidoglycan biosynthesis is an ideal site at which to look for novel antimicrobial targets. Phage display was used to develop novel peptide inhibitors for MurC, an essential enzyme involved in the early steps of biosynthesis of peptidoglycan monomer.
Methods: We cloned and overexpressed the murA, -B and -C genes from Pseudomonas aeruginosa in the pET expression vector, adding a His-tag to their C termini. The three proteins were overproduced in Escherichia coli and purified to homogeneity in milligram quantities. MurA and -B were combinatorially used to synthesize the MurC substrate UDP-N-acetylmuramate, the identity of which was confirmed by mass spectrometry and nuclear magnetic resonance analysis. Two phage-display libraries were screened against MurC in order to identify peptide ligands to the enzyme.
Results: Three rounds of biopanning were carried out, successively increasing elution specificity from round 1 to 3. The third round was accomplished with both non-specific elution and competitive elution with each of the three MurC substrates, UDP-N-acetylmuramic acid (UNAM), ATP and L-alanine. The DNA of 10 phage, selected randomly from each group, was extracted and sequenced, and consensus peptide sequences were elucidated. Peptides were synthesized and tested for inhibition of the MurC-catalysed reaction, and two peptides were shown to be inhibitors of MurC activity with IC50s of 1.5 and 0.9 mM, respectively.
Conclusion: The powerful selection technique of phage display allowed us to identify two peptide inhibitors of the essential bacterial enzyme MurC. The peptide sequences represent the basis for the synthesis of inhibitory peptidomimetic molecules.
Keywords: MurC, peptide inhibitors, phage display
* Corresponding author. Tel: +1-418-656-3070; Fax: +1-418-656-7176; E-mail: rclevesq{at}rsvs.ulaval.ca
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