Abstract
BackgroundTo 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.ResultsScreening 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.ConclusionMurFp1 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.
Highlights
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
N-terminal sequencing of the first 15 amino acid residues confirmed its identity as P. aeruginosa MurF ligase [Swiss-Prot: Q9EY48, PIR: SF001562] (100% identical to the published sequence, including the initial Met)
As a first step into the development of novel inhibitors targeting unexploited bacterial targets, we have identified the first peptide inhibitor of the essential MurF amide ligase enzyme using a phage display approach
Summary
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. The bacterial cell wall biosynthesis pathway represents the most validated source of antibacterial targets. The pathway encodes essential and highly conserved enzymes with no eukaryotic counterparts, the inhibition of which leading to bacterial cell death [1]. Membrane translocases MraY and MurG add the undecaprenyl-phosphate lipid carrier and N-acetylglucosamine to form lipid II. This precursor is translocated to the periplasm and linked to the growing cell wall polymer by the transglycosylation and transpeptidation actions of penicillin-binding proteins (PBPs). The cell wall layer, composed of alternating units of UDP-N-acetylglucosamine and UDP-N-acetylmuramic acid cross-linked via short peptide chains (Figure 1), maintains cell shape and integrity [2]
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