JAC Advance Access published online on January 28, 2003
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkg010
© 2003 by The British Society for Antimicrobial Chemotherapy
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Original article
1 Faculté de Médecine, 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
* Corresponding author. E-mail: rclevesq{at}rsvs.ulaval.ca.
Received 14 June 2002
; revised 13 August 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
Identification of novel inhibitors of Pseudomonas
aeruginosa MurC enzyme derived from phage-displayed peptide
libraries
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
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