Identification of novel inhibitors of Pseudomonas aeruginosa MurC enzyme derived from phage-displayed peptide libraries.
OBJECTIVE
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 IC(50)s of 1.5 and 0.9 mM, respectively.


CONCLUSIONS
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.
El Zoeiby
Ahmed
A
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.
Sanschagrin
François
F
Darveau
André
A
Brisson
Jean-Robert
JR
Levesque
Roger C
RC
D000595:Amino Acid Sequence; D017104:Bacteriophage M13; D016503:Drug Delivery Systems / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000821:virology; D008969:Molecular Sequence Data; D019151:Peptide Library; D010453:Peptide Synthases / Q000037:antagonists & inhibitors* / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000235:genetics; D020539:Sequence Analysis, Protein / Q000379:methods*
D004791:Enzyme Inhibitors; D019151:Peptide Library; D010453:Peptide Synthases; C023229:UDP-N-acetylmuramoyl-alanine synthetase