The Library

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

Tools

Paradis-Bleau , Catherine, Lloyd, Adrian, Sanschagrin, François, Clark, Tom, Blewett, Ann, Bugg, Tim and Levesque, Roger C. . (2008) Phage display-derived inhibitor of the essential cell wall biosynthesis enzyme MurF. BMC Biochemistry, Vol.9 (No.33). ISSN 1471-2091

Preview

PDF
WRAP_Lloyd_1471_2091_9_33.pdf - Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader
Download (506Kb)

Official URL: http://dx.doi.org/10.1186/1471-2091-9-33

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

Item Type:	Journal Article
Subjects:	Q Science > QH Natural history > QH301 Biology
Divisions:	Faculty of Science > Life Sciences (2010- ) > Biological Sciences ( -2010) Faculty of Science > Chemistry
Library of Congress Subject Headings (LCSH):	Antibacterial agents
Journal or Publication Title:	BMC Biochemistry
Publisher:	BioMed Central Ltd.
ISSN:	1471-2091
Date:	19 December 2008
Volume:	Vol.9
Number:	No.33
Identification Number:	10.1186/1471-2091-9-33
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Open Access
Funder:	Networks of Centres of Excellence (Canada), Fonds FCAR (Québec), Fonds de la recherche en santé du Québec (FRSQ)
References:	1. Projan SJ: New (and not so new) antibacterial targets – from where and when will the novel drugs come? Curr Opin Pharmacol 2002, 2:513-522. 2. Young KD: Bacterial shape. Mol Microbiol 2003, 49:571-580. 3. El Zoeiby A, Sanschagrin F, Levesque RC: Structure and function of the Mur enzymes: development of novel inhibitors. Mol Microbiol 2003, 47:1-12. 4. Silver LL: Novel inhibitors of bacterial cell wall synthesis. Curr Opin Microbiol 2003, 6:431-438. 5. Bugg TD, Walsh CT: Intracellular steps of bacterial cell wall peptidoglycan biosynthesis: enzymology, antibiotics, and antibiotic resistance. Nat Prod Rep 1992, 9:199-215. 6. Anderson MS, Eveland SS, Onishi HR, Pompliano DL: Kinetic mechanism of the Escherichia coli UDPMurNAc-tripeptide D-alanyl- D-alanine-adding enzyme: use of a glutathione Stransferase fusion. Biochemistry 1996, 35:16264-16269. 7. Mengin-Lecreulx D, van Heijenoort J, Park 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. J Bacteriol 1996, 178:5347-5352. 8. Lugtenberg EJ, 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. J Bacteriol 1972, 110:35-40. 9. Sobral RG, Ludovice AM, de Lencastre H, Tomasz A: Role of murF in cell wall biosynthesis: isolation and characterization of a murF conditional mutant of Staphylococcus aureus. J Bacteriol 2006, 188:2543-2553. 10. Baum EZ, Crespo-Carbone SM, Abbanat D, Foleno B, Maden A, Goldschmidt R, Bush K: Utility of muropeptide ligase for identification of inhibitors of the cell wall biosynthesis enzyme MurF. Antimicrob Agents Chemother 2006, 50:230-236. 11. Walsh CT: Enzymes in the D-alanine branch of bacterial cell wall peptidoglycan assembly. J Biol Chem 1989, 264:2393-2396. 12. Holtje JV: Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli. Microbiol Mol Biol Rev 1998, 62:181-203. 13. Sobral RG, Ludovice AM, Gardete S, Tabei K, De Lencastre H, Tomasz A: Normally functioning murF is essential for the optimal expression of methicillin resistance in Staphylococcus aureus. Microb Drug Resist 2003, 9:231-241. 14. Bugg TD, Wright GD, Dutka-Malen S, Arthur M, Courvalin P, Walsh CT: Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA. Biochemistry 1991, 30:10408-10415. 15. Reynolds PE: Structure, biochemistry and mechanism of action of glycopeptide antibiotics. Eur J Clin Microbiol Infect Dis 1989, 8:943-950. 16. Christensen DJ, Gottlin EB, Benson RE, Hamilton PT: Phage display for target-based antibacterial drug discovery. Drug Discov Today 2001, 6:721-727. 17. El Zoeiby A, Sanschagrin F, Darveau A, Brisson JR, Levesque RC: Identification of novel inhibitors of Pseudomonas aeruginosa MurC enzyme derived from phage-displayed peptide libraries. J Antimicrob Chemother 2003, 51:531-543. 18. Paradis-Bleau C, Beaumont M, Boudreault L, Lloyd A, Sanschagrin F, Bugg TD, Levesque RC: Selection of peptide inhibitors against the Pseudomonas aeruginosa MurD cell wall enzyme. Peptides 2006. 19. Davies JC: Pseudomonas aeruginosa in cystic fibrosis: pathogenesis and persistence. Paediatr Respir Rev 2002, 3:128-134. 20. Pierce GE: Pseudomonas aeruginosa, Candida albicans, and device-related nosocomial infections: implications, trends, and potential approaches for control. J Ind Microbiol Biotechnol 2005, 32:309-318. 21. El Zoeiby A, Sanschagrin F, Havugimana PC, Garnier A, Levesque RC: In vitro reconstruction of the biosynthetic pathway of peptidoglycan cytoplasmic precursor in Pseudomonas aeruginosa. FEMS Microbiol Lett 2001, 201:229-235. 22. Comb DG: The enzymatic addition of D-alanyl-D-alanine to a uridine nucleotide-peptide. J Biol Chem 1962, 237:1601-1604. 23. Paradis-Bleau C, Sanschagrin F, Levesque RC: Peptide inhibitors of the essential cell division protein FtsA. Protein Eng Des Sel 2005, 18:85-91. 24. Paradis-Bleau C, Sanschagrin F, Levesque RC: Identification of Pseudomonas aeruginosa FtsZ peptide inhibitors as a tool for development of novel antimicrobials. J Antimicrob Chemother 2004, 54:278-280. 25. Egan A, Lawrence P, Strominger JL: Enzymatic synthesis of the peptide in bacterial ridine nucleotides. V. Co ++ -dependent reversal of peptide bond formation. J Biol Chem 1973, 248:3122-3130. 26. Neuhaus FC, Struve 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 1965, 4:120-131. 27. Michaud C, Blanot D, Flouret B, Van Heijenoort J: Partial purification and specificity studies of the D-glutamate-adding and Dalanyl- D-alanine-adding enzymes from Escherichia coli K12. Eur J Biochem 1987, 166:631-637. 28. Dementin S, Bouhss A, Auger G, Parquet C, Mengin-Lecreulx D, Dideberg O, van Heijenoort J, Blanot D: Evidence of a functional requirement for a carbamoylated lysine residue in MurD, MurE and MurF synthetases as established by chemical rescue experiments. Eur J Biochem 2001, 268:5800-5807. 29. Mengin-Lecreulx D, Flouret B, van Heijenoort J: Cytoplasmic steps of peptidoglycan synthesis in Escherichia coli. J Bacteriol 1982, 151:1109-1117. 30. Mengin-Lecreulx D, Blanot D, van Heijenoort J: Replacement of diaminopimelic acid by cystathionine or lanthionine in the peptidoglycan of Escherichia coli. J Bacteriol 1994, 176:4321-4327. 31. Lugtenberg EJ: Studies on Escherichia coli enzymes involved in the synthesis of uridine diphosphate-N-acetyl-muramyl-pentapeptide. J Bacteriol 1972, 110:26-34. 32. Duncan K, van Heijenoort J, Walsh CT: Purification and characterization of the D-alanyl-D-alanine-adding enzyme from Escherichia coli. Biochemistry 1990, 29:2379-2386. 33. Eveland SS, Pompliano DL, Anderson 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 1997, 36:6223-6229. 34. Azzolina BA, Yuan X, Anderson MS, El-Sherbeini M: 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 2001, 21:393-400. 35. Gu YG, Florjancic AS, Clark RF, Zhang T, Cooper CS, Anderson DD, Lerner CG, McCall JO, Cai Y, Black-Schaefer CL, Stamper GF, Hajduk PJ, Beutel BA: Structure-activity relationships of novel potent MurF inhibitors. Bioorg Med Chem Lett 2004, 14:267-270. 36. Comess KM, Schurdak ME, Voorbach MJ, Coen M, Trumbull JD, Yang H, Gao L, Tang H, Cheng X, Lerner CG, McCall JO, Burns DJ, Beutel BA: An ultraefficient affinity-based high-throughout screening process: application to bacterial cell wall biosynthesis enzyme MurF. J Biomol Screen 2006, 11:743-754. 37. Miller DJ, Hammond SM, Anderluzzi D, Bugg TDH: Aminoalkylphosphinate inhibitors of D-Ala-D-Ala adding enzyme. J Chem Soc Perkin Trans 1998, 1:131-142. 38. Bouhss A, Mengin-Lecreulx D, Blanot D, van Heijenoort J, 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 1997, 36:11556-11563. 39. Bertrand JA, Fanchon E, Martin L, Chantalat L, Auger G, Blanot D, van Heijenoort J, Dideberg O: "Open" structures of MurD: domain movements and structural similarities with folylpolyglutamate synthetase. J Mol Biol 2000, 301:1257-1266. 40. Yan Y, Munshi S, Leiting B, Anderson MS, Chrzas J, Chen Z: Crystal structure of Escherichia coli UDPMurNAc-tripeptide d-alanyl- d-alanine-adding enzyme (MurF) at 2.3 A resolution. J Mol Biol 2000, 304:435-445. 41. Cornish-Bowden A: Fundamentals of enzyme kinetics London; Boston: Butterworths; 1979. 42. Segal IH: Enzyme kinetics – behavior and analysis of rapid equilibrium and steady-state enzyme systems John Wiley and Sons, Inc; 1993. 43. Nefzi A, Dooley C, Ostresh JM, Houghten RA: Combinatorial chemistry: from peptides and peptidomimetics to small organic and heterocyclic compounds. Bioorg Med Chem Lett 1998, 8:2273-2278. 44. Walker JM: The proteomics protocols handbook Totowa, N.J.: Humana Press; 2005. 45. Jones DT: Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 1999, 292:195-202. 46. Mackey AJ, Haystead TA, Pearson WR: Getting more from less: algorithms for rapid protein identification with multiple short peptide sequences. Mol Cell Proteomics 2002, 1:139-147. 47. Reddy SG, Waddell ST, Kuo DW, Wong KK, Pompliano DL: Preparative Enzymatic Synthesis and Characterization of the Cytoplasmic Intermediates of Murein Biosynthesis. Journal of the American Chemical Society 1999, 121:1175-1178. 48. Lanzetta PA, Alvarez LJ, Reinach PS, Candia OA: An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem 1979, 100:95-97.
URI:	http://wrap.warwick.ac.uk/id/eprint/411