Phage display-derived inhibitor of the essential cell wall biosynthesis enzyme MurF
Paradis-Bleau , Catherine, Lloyd, Adrian, Sanschagrin, François, Clark, Tom, Blewett, Ann, Bugg, Tim and Levesque, Roger C. . (2008) Phage display-derived inhibitor of the essential cell wall biosynthesis enzyme MurF. BMC Biochemistry, Vol.9 (No.33). ISSN 1471-2091
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Official URL: http://dx.doi.org/10.1186/1471-2091-9-33
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.
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 Ki was established at 420 μM with respect to the mixed type of inhibition against D-Ala-D-Ala.
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.
|Item Type:||Journal Article|
|Subjects:||Q Science > QH Natural history > QH301 Biology|
|Divisions:||Faculty of Science > Life Sciences (2010- ) > Biological Sciences ( -2010)
Faculty of Science > Chemistry
|Library of Congress Subject Headings (LCSH):||Antibacterial agents|
|Journal or Publication Title:||BMC Biochemistry|
|Publisher:||BioMed Central Ltd.|
|Official Date:||19 December 2008|
|Access rights to Published version:||Open Access|
|Funder:||Networks of Centres of Excellence (Canada), Fonds FCAR (Québec), Fonds de la recherche en santé du Québec (FRSQ)|
1. Projan SJ: New (and not so new) antibacterial targets – from
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