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

Paradis-Bleau C, Lloyd A, Sanschagrin F, Clarke T, Blewett A, Bugg TD, Levesque RC - BMC Biochem. (2008)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Département de Biologie Médicale, Université Laval, Sainte-Foy, Québec G1K 7P4, Canada. Catherine_Paradis-Bleau@hms.harvard.edu

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 ified 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.

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.

Show MeSH
Inhibition of MurF activity by MurFp1 as a function of pre-incubation time. Experiments were done following different pre-incubation times of MurF with 2 mM of MurFp1.
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Figure 3: Inhibition of MurF activity by MurFp1 as a function of pre-incubation time. Experiments were done following different pre-incubation times of MurF with 2 mM of MurFp1.

Mentions: MurFp1 was first shown to be a weak time-dependent inhibitor of MurF. The inhibition increased as a function of pre-incubation time, following an overall linear relationship. At a concentration of 2 mM, MurFp1 inhibited 50% of MurF activity subsequent to a 30-minute pre-incubation time (Figure 3).


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

Paradis-Bleau C, Lloyd A, Sanschagrin F, Clarke T, Blewett A, Bugg TD, Levesque RC - BMC Biochem. (2008)

Inhibition of MurF activity by MurFp1 as a function of pre-incubation time. Experiments were done following different pre-incubation times of MurF with 2 mM of MurFp1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2626591&req=5

Figure 3: Inhibition of MurF activity by MurFp1 as a function of pre-incubation time. Experiments were done following different pre-incubation times of MurF with 2 mM of MurFp1.
Mentions: MurFp1 was first shown to be a weak time-dependent inhibitor of MurF. The inhibition increased as a function of pre-incubation time, following an overall linear relationship. At a concentration of 2 mM, MurFp1 inhibited 50% of MurF activity subsequent to a 30-minute pre-incubation time (Figure 3).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Département de Biologie Médicale, Université Laval, Sainte-Foy, Québec G1K 7P4, Canada. Catherine_Paradis-Bleau@hms.harvard.edu

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 ified 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.

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.

Show MeSH