Skip to main content
Article thumbnail
Location of Repository

Phage display-derived inhibitor of the essential cell wall biosynthesis enzyme MurF

By Catherine Paradis-Bleau, Adrian Lloyd, François Sanschagrin, Tom Clark, Ann Blewett, Tim Bugg and Roger C. Levesque

Abstract

Background\ud 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.\ud \ud Results\ud 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 μM, and the Ki was established at 420 μM with respect to the mixed type of inhibition against D-Ala-D-Ala.\ud \ud Conclusion\ud 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.\ud \u

Topics: QH301
Publisher: BioMed Central Ltd.
Year: 2008
OAI identifier: oai:wrap.warwick.ac.uk:411

Suggested articles

Citations

  1. (2006). An ultraefficient affinity-based high-throughout screening process: application to bacterial cell wall biosynthesis enzyme MurF. doi
  2. (2003). Bacterial shape. Mol Microbiol
  3. (2004). Beutel BA: Structure-activity relationships of novel potent MurF inhibitors. Bioorg Med Chem Lett doi
  4. (2001). Blanot D: Evidence of a functional requirement for a carbamoylated lysine residue in MurD, MurE and MurF synthetases as established by chemical rescue experiments. doi
  5. (1992). CT: Intracellular steps of bacterial cell wall peptidoglycan biosynthesis: enzymology, antibiotics, and antibiotic resistance. Nat Prod Rep doi
  6. (1991). CT: Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA. Biochemistry doi
  7. (1990). CT: Purification and characterization of the D-alanyl-D-alanine-adding enzyme from Escherichia coli. Biochemistry doi
  8. (1982). Cytoplasmic steps of peptidoglycan synthesis in Escherichia coli. doi
  9. (2000). Dideberg O: "Open" structures of MurD: domain movements and structural similarities with folylpolyglutamate synthetase. doi
  10. (1993). Enzyme kinetics – behavior and analysis of rapid equilibrium and steady-state enzyme systems doi
  11. (1989). Enzymes in the D-alanine branch of bacterial cell wall peptidoglycan assembly.
  12. (1979). Fundamentals of enzyme kinetics London; doi
  13. (1998). Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli. Microbiol Mol Biol Rev
  14. (1998). Houghten RA: Combinatorial chemistry: from peptides and peptidomimetics to small organic and heterocyclic compounds. Bioorg Med Chem Lett doi
  15. (1973). JL: Enzymatic synthesis of the peptide in bacterial ridine nucleotides. V. Co ++ -dependent reversal of peptide bond formation.
  16. (1996). JT: Identification of the mpl gene encoding UDP-N-acetylmuramate: L-alanylgamma-D-glutamyl-meso-diaminopimelate ligase in Escherichia coli and its role in recycling of cell wall peptidoglycan.
  17. (2001). Levesque RC: In vitro reconstruction of the biosynthetic pathway of peptidoglycan cytoplasmic precursor in Pseudomonas aeruginosa. doi
  18. (2006). Levesque RC: Selection of peptide inhibitors against the Pseudomonas aeruginosa MurD cell wall enzyme. Peptides doi
  19. (1997). MS: Conditionally lethal Escherichia coli murein mutants contain point defects that map to regions conserved among murein and folyl polygamma-glutamate ligases: identification of a ligase superfamily. Biochemistry doi
  20. (2002). New (and not so new) antibacterial targets – from where and when will the novel drugs come? Curr Opin Pharmacol doi
  21. (2003). Novel inhibitors of bacterial cell wall synthesis. Curr Opin Microbiol
  22. (1979). OA: An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem doi
  23. (1997). Parquet C: Invariant amino acids in the Mur peptide synthetases of bacterial peptidoglycan synthesis and their modification by sitedirected mutagenesis in the UDP-MurNAc:L-alanine ligase from Escherichia coli. Biochemistry doi
  24. (1987). Partial purification and specificity studies of the D-glutamate-adding and Dalanyl-D-alanine-adding enzymes from Escherichia coli K12. doi
  25. (2002). Pearson WR: Getting more from less: algorithms for rapid protein identification with multiple short peptide sequences. Mol Cell Proteomics doi
  26. (1996). Pompliano DL: Kinetic mechanism of the Escherichia coli UDPMurNAc-tripeptide D-alanyl-D-alanine-adding enzyme: use of a glutathione Stransferase fusion. Biochemistry doi
  27. (1999). Pompliano DL: Preparative Enzymatic Synthesis and Characterization of the Cytoplasmic Intermediates of Murein Biosynthesis. doi
  28. (1999). Protein secondary structure prediction based on position-specific scoring matrices. doi
  29. (2002). Pseudomonas aeruginosa in cystic fibrosis: pathogenesis and persistence. Paediatr Respir Rev doi
  30. (2005). Pseudomonas aeruginosa, Candida albicans, and device-related nosocomial infections: implications, trends, and potential approaches for control. doi
  31. (2001). PT: Phage display for target-based antibacterial drug discovery. Drug Discov Today doi
  32. (2003). RC: Identification of novel inhibitors of Pseudomonas aeruginosa MurC enzyme derived from phage-displayed peptide libraries. doi
  33. (2004). RC: Identification of Pseudomonas aeruginosa FtsZ peptide inhibitors as a tool for development of novel antimicrobials. doi
  34. (2005). RC: Peptide inhibitors of the essential cell division protein FtsA. Protein Eng Des Sel doi
  35. (2003). RC: Structure and function of the Mur enzymes: development of novel inhibitors. Mol Microbiol doi
  36. (1994). Replacement of diaminopimelic acid by cystathionine or lanthionine in the peptidoglycan of Escherichia coli.
  37. (1989). Structure, biochemistry and mechanism of action of glycopeptide antibiotics. doi
  38. (1972). Studies on Escherichia coli enzymes involved in the synthesis of uridine diphosphate-N-acetyl-muramyl-pentapeptide.
  39. (1998). TDH: Aminoalkylphosphinate inhibitors of D-Ala-D-Ala adding enzyme. J Chem Soc Perkin Trans doi
  40. (2001). The cell wall and cell division gene cluster in the Mra operon of Pseudomonas aeruginosa: cloning, production, and purification of active enzymes. Protein Expr Purif doi
  41. (1962). The enzymatic addition of D-alanyl-D-alanine to a uridine nucleotide-peptide.
  42. (2005). The proteomics protocols handbook doi
  43. (2003). Tomasz A: Normally functioning murF is essential for the optimal expression of methicillin resistance in Staphylococcus aureus. Microb Drug Resist doi
  44. (2006). Tomasz A: Role of murF in cell wall biosynthesis: isolation and characterization of a murF conditional mutant of Staphylococcus aureus. doi
  45. (2006). Utility of muropeptide ligase for identification of inhibitors of the cell wall biosynthesis enzyme MurF. Antimicrob Agents Chemother doi
  46. (1972). v Schijndel-van Dam A: Temperature-sensitive mutants of Escherichia coli K-12 with low activities of the Lalanine adding enzyme and the D-alanyl-D-alanine adding enzyme.
  47. (1965). WG: Enzymatic Synthesis of Analogs of the Cell-Wall Precursor. I. Kinetics and Specificity of Uridine Diphospho-N-Acetylmuramyl-L-Alanyl-D-Glutamyl-LLysine:D-Alanyl-D-Alanine Ligase (Adenosine Diphosphate) from Streptococcus Faecalis R. Biochemistry doi

To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.