Research article

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

Catherine Paradis-Bleau¹ , Adrian Lloyd² , François Sanschagrin¹ , Tom Clarke² , Ann Blewett² , Timothy DH Bugg² and Roger C Levesque¹

¹  Département de Biologie Médicale, Université Laval, Sainte-Foy, Québec G1K 7P4 , Canada

²  Department of chemistry, University of Warwick, Coventry, CV4 7AL, United Kingdom

author email corresponding author email

BMC Biochemistry 2008, 9:33doi:10.1186/1471-2091-9-33

Published:	19 December 2008

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 K_i 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.