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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.

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IC50 determination for the inhibition of MurF activity by MurFp1. Experiments were done following a 30 min of pre-incubation step with MurF, MurFp1 and UDP-MurNAc-Ala-Glu-A2pm.
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Figure 4: IC50 determination for the inhibition of MurF activity by MurFp1. Experiments were done following a 30 min of pre-incubation step with MurF, MurFp1 and UDP-MurNAc-Ala-Glu-A2pm.

Mentions: Adding UDP-MurNAc-Ala-Glu-meso-A2pm or ATP during the pre-incubation step unexpectedly increased the inhibitory action of MurFp1, an effect less pronounced for ATP (data not shown). MurF reaction velocity in the absence of inhibitor was similar with or without UDP-MurNAc-Ala-Glu-meso-A2pm or ATP in the pre-incubation step. MurFp1 inhibited MurF eight times more efficiently when UDP-MurNAc-Ala-Glu-A2pm was added to the 30-minute pre-incubation step, giving an IC50 value of 250 ± 10 μM. The inhibition curve displayed a linear dose-response trend having variable slopes as shown in Figure 4.


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)

IC50 determination for the inhibition of MurF activity by MurFp1. Experiments were done following a 30 min of pre-incubation step with MurF, MurFp1 and UDP-MurNAc-Ala-Glu-A2pm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: IC50 determination for the inhibition of MurF activity by MurFp1. Experiments were done following a 30 min of pre-incubation step with MurF, MurFp1 and UDP-MurNAc-Ala-Glu-A2pm.
Mentions: Adding UDP-MurNAc-Ala-Glu-meso-A2pm or ATP during the pre-incubation step unexpectedly increased the inhibitory action of MurFp1, an effect less pronounced for ATP (data not shown). MurF reaction velocity in the absence of inhibitor was similar with or without UDP-MurNAc-Ala-Glu-meso-A2pm or ATP in the pre-incubation step. MurFp1 inhibited MurF eight times more efficiently when UDP-MurNAc-Ala-Glu-A2pm was added to the 30-minute pre-incubation step, giving an IC50 value of 250 ± 10 μM. The inhibition curve displayed a linear dose-response trend having variable slopes as shown in Figure 4.

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