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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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Kinetics of MurF inhibition by MurFp1 with respect to D-Ala-D-Ala. A) Michaelis-Menten and B) Lineweaver-Burk plots for MurF activity with respect to the D-Ala-D-Ala substrate showing inhibition by MurFp1 at 0 μM (●), 75 μM (■), 150 μM (▲), 300 μM (▼) and 600 μM (◆).
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Figure 5: Kinetics of MurF inhibition by MurFp1 with respect to D-Ala-D-Ala. A) Michaelis-Menten and B) Lineweaver-Burk plots for MurF activity with respect to the D-Ala-D-Ala substrate showing inhibition by MurFp1 at 0 μM (●), 75 μM (■), 150 μM (▲), 300 μM (▼) and 600 μM (◆).

Mentions: The presence of D-Ala-D-Ala during the pre-incubation step ified the inhibition of MurF by MurFp1 (data not shown). MurF reaction velocity in the absence of inhibitor was nearly identical with or without D-Ala-D-Ala in the pre-incubation step. The Ki of MurFp1 was determined with respect to the D-Ala-D-Ala substrate, with a 30 min pre-incubation of MurF with UDP-MurNAc-Ala-Glu-A2pm and MurFp1. The Km values of MurF for D-Ala-D-Ala remained almost the same for each MurFp1 concentration used to determine the Ki while the Vmax of MurF decreased significantly as a function of MurFp1 concentration (Figure 5A and Table 3). At 600 μM MurFp1, the Vmax of MurF was three times lower than for the uninhibited reaction (Table 3). The slopes and the y-intercept of the fitted lines of the Lineweaver-Burk plot varied with MurFp1 concentration (Figure 5B), indicating a catalytic and specific component in the inhibition. The common intersection point of the fitted lines of the Lineweaver-Burk plot was positioned at the left of the ordinate and below the abscissa (Figure 5B), indicating a reversible mixed type of inhibition against D-Ala-D-Ala. This inhibition type was further identified as the most suitable model of inhibition, based on statistical convergence to the experimental data according to the Runs Test, giving the highest R2 value (0.97), the lowest Akaike value (-275) and the lowest standard deviation of the residuals (0.15). The non-competitive type of inhibition was the second best model, giving statistical values close to the mixed model of inhibition (data not shown). The Ki value of MurFp1 was 420 ± 100 μM for the mixed type of inhibition against D-Ala-D-Ala, and 370 ± 55 μM for the non-competitive model. The a parameter indicated that MurFp1 binding to the MurF-D-Ala-D-Ala complex (ES) did not significantly change MurF affinity for D-Ala-D-Ala. The β parameter indicated that the rate constant of ES breaking down to enzyme and product dropped when MurFp1 was bound to ES (Table 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)

Kinetics of MurF inhibition by MurFp1 with respect to D-Ala-D-Ala. A) Michaelis-Menten and B) Lineweaver-Burk plots for MurF activity with respect to the D-Ala-D-Ala substrate showing inhibition by MurFp1 at 0 μM (●), 75 μM (■), 150 μM (▲), 300 μM (▼) and 600 μM (◆).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Kinetics of MurF inhibition by MurFp1 with respect to D-Ala-D-Ala. A) Michaelis-Menten and B) Lineweaver-Burk plots for MurF activity with respect to the D-Ala-D-Ala substrate showing inhibition by MurFp1 at 0 μM (●), 75 μM (■), 150 μM (▲), 300 μM (▼) and 600 μM (◆).
Mentions: The presence of D-Ala-D-Ala during the pre-incubation step ified the inhibition of MurF by MurFp1 (data not shown). MurF reaction velocity in the absence of inhibitor was nearly identical with or without D-Ala-D-Ala in the pre-incubation step. The Ki of MurFp1 was determined with respect to the D-Ala-D-Ala substrate, with a 30 min pre-incubation of MurF with UDP-MurNAc-Ala-Glu-A2pm and MurFp1. The Km values of MurF for D-Ala-D-Ala remained almost the same for each MurFp1 concentration used to determine the Ki while the Vmax of MurF decreased significantly as a function of MurFp1 concentration (Figure 5A and Table 3). At 600 μM MurFp1, the Vmax of MurF was three times lower than for the uninhibited reaction (Table 3). The slopes and the y-intercept of the fitted lines of the Lineweaver-Burk plot varied with MurFp1 concentration (Figure 5B), indicating a catalytic and specific component in the inhibition. The common intersection point of the fitted lines of the Lineweaver-Burk plot was positioned at the left of the ordinate and below the abscissa (Figure 5B), indicating a reversible mixed type of inhibition against D-Ala-D-Ala. This inhibition type was further identified as the most suitable model of inhibition, based on statistical convergence to the experimental data according to the Runs Test, giving the highest R2 value (0.97), the lowest Akaike value (-275) and the lowest standard deviation of the residuals (0.15). The non-competitive type of inhibition was the second best model, giving statistical values close to the mixed model of inhibition (data not shown). The Ki value of MurFp1 was 420 ± 100 μM for the mixed type of inhibition against D-Ala-D-Ala, and 370 ± 55 μM for the non-competitive model. The a parameter indicated that MurFp1 binding to the MurF-D-Ala-D-Ala complex (ES) did not significantly change MurF affinity for D-Ala-D-Ala. The β parameter indicated that the rate constant of ES breaking down to enzyme and product dropped when MurFp1 was bound to ES (Table 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.

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