Selection of peptide inhibitors against the Pseudomonas aeruginosa MurD cell wall enzyme.
The purified Pseudomonas aeruginosa cell wall biosynthesis MurD amide ligase enzyme was used to screen C-7-C and 12 mers peptides from phage display libraries using competitive biopanning approaches with the specific substrates D-glutamate and ATP. From the 60 phage-encoded peptides identified, DNA was sequenced, deduced amino acid sequences aligned and two peptides were synthesized from consensus sequences identified. The UDP-N-acetylmuramyl-L-alanine MurD substrate was synthesized, purified and used to develop a spectrophotometric assay. One peptide synthesized was found to specifically inhibit ATPase activity of MurD. The IC50 value was estimated at 4 microM for the C-7-C MurDp1 peptide. The loop conformation of MurDp1 was shown to be important for the inhibition of the UDP-N-acetylmuramyl-L-alanine:D-glutamate MurD ligase. The linear 12 mers MurD2 peptide has an IC50 value of 15 mM. A conserved amino acid motif was found between MurDp2 and the bacterial glyceraldehyde 3-phosphate dehydrogenase indicating that MurDp2 binds at a protein-protein interacting site. The approach proposed and results obtained suggest that efficient peptide inhibitors as well as protein-protein interaction domains can be identified by phage display.
Paradis-Bleau
Catherine
C
CREFSIP, Département de Biologie Médicale, Faculté de Médecine, Université Laval, Sainte-Foy, Québec, Canada G1K 7P4. cparadis@rsvs.ulval.ca
Beaumont
Mélanie
M
Boudreault
Lydia
L
Lloyd
Adrian
A
Sanschagrin
François
F
Bugg
Timothy D H
TD
Levesque
Roger C
RC
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D019295:Computational Biology / Q000379:methods; D004305:Dose-Response Relationship, Drug; D020128:Inhibitory Concentration 50; D008025:Ligases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry
D019151:Peptide Library; D010455:Peptides; D008025:Ligases