title: Phage display-derived inhibitor of the essential cell wall biosynthesis enzyme MurF.
abstract: BACKGROUND
  To develop antibacterial agents having novel modes of action against bacterial cell wall biosynthesis, we targeted the essential MurF enzyme of the antibiotic resistant pathogen Pseudomonas aeruginosa. MurF catalyzes the formation of a peptide bond between D-Alanyl-D-Alanine (D-Ala-D-Ala) and the cell wall precursor uridine 5'-diphosphoryl N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid (UDP-MurNAc-Ala-Glu-meso-A2pm) with the concomitant hydrolysis of ATP to ADP and inorganic phosphate, yielding UDP-N-acetylmuramyl-pentapeptide. As MurF acts on a dipeptide, we exploited a phage display approach to identify peptide ligands having high binding affinities for the enzyme.
  
  
  RESULTS
  Screening of a phage display 12-mer library using purified P. aeruginosa MurF yielded to the identification of the MurFp1 peptide. The MurF substrate UDP-MurNAc-Ala-Glumeso-A2pm was synthesized and used to develop a sensitive spectrophotometric assay to quantify MurF kinetics and inhibition. MurFp1 acted as a weak, time-dependent inhibitor of MurF activity but was a potent inhibitor when MurF was pre-incubated with UDP-MurNAc-Ala-Glu-meso-A2pm or ATP. In contrast, adding the substrate D-Ala-D-Ala during the pre-incubation nullified the inhibition. The IC50 value of MurFp1 was evaluated at 250 microM, and the Ki was established at 420 microM with respect to the mixed type of inhibition against D-Ala-D-Ala.
  
  
  CONCLUSIONS
  MurFp1 exerts its inhibitory action by interfering with the utilization of D-Ala-D-Ala by the MurF amide ligase enzyme. We propose that MurFp1 exploits UDP-MurNAc-Ala-Glu-meso-A2pm-induced structural changes for better interaction with the enzyme. We present the first peptide inhibitor of MurF, an enzyme that should be exploited as a target for antimicrobial drug development.
authors.affiliation: Département de Biologie Médicale, Université Laval, Sainte-Foy, Québec G1K 7P4, Canada. Catherine_Paradis-Bleau@hms.harvard.edu
authors.forename: Catherine
authors.initials: C
authors.lastname: Paradis-Bleau
authors.forename: Adrian
authors.initials: A
authors.lastname: Lloyd
authors.forename: François
authors.initials: F
authors.lastname: Sanschagrin
authors.forename: Tom
authors.initials: T
authors.lastname: Clarke
authors.forename: Ann
authors.initials: A
authors.lastname: Blewett
authors.forename: Timothy D H
authors.initials: TD
authors.lastname: Bugg
authors.forename: Roger C
authors.initials: RC
authors.lastname: Levesque
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D053898:Biosynthetic Pathways; D002473:Cell Wall / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007700:Kinetics; D008969:Molecular Sequence Data; D019151:Peptide Library; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D019151:Peptide Library