A peptide inhibitor of MurA UDP-N-acetylglucosamine enolpyruvyl transferase: the first committed step in peptidoglycan biosynthesis.
The MurA enzyme from Pseudomonas aeruginosa was purified to homogeneity and found to be biologically active as a UDP-N-acetylglucosamine (UNAG) enolpyruvyl transferase in a coupled enzyme assay where ATPase activity was measured by the release of inorganic phosphate. A microtiter plate assay coupled to competitive biopanning using the UDP-N-acetylglucosamine was used to screen 10(9) C-7-C and 12-mers peptides from phage display libraries. From 60 phage-encoded peptides identified after the fourth round of biopanning, deduced amino acid sequences were aligned and two peptides were synthesized and tested for inhibition of the MurA-catalyzed reaction. The PEP 1354 peptide inhibited the ATPase activity of MurA with an IC(50) value of 200muM and was found to be a competitive inhibitor of UNAG. The pre-incubation of MurA with inhibitor indicated a time-independent inhibition. This time-dependent inhibition is the first report of peptide inhibitors of MurA, which represent the scaffold for the synthesis of inhibitory peptidomimetic molecules.
Molina-López
José
J
Departamento de Salud Pública, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico. joseml@servidor.unam.mx
Sanschagrin
François
F
Levesque
Roger C
RC
D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D010455:Peptides / Q000502:physiology*; D010457:Peptidoglycan / Q000096:biosynthesis*; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D014539:Uridine Diphosphate Sugars / Q000378:metabolism*
D010455:Peptides; D010457:Peptidoglycan; D014539:Uridine Diphosphate Sugars; C083732:uridine diphosphate N-acetyllactosamine; D019883:Alkyl and Aryl Transferases; C022566:UDP-N-acetylglucosamine 1-carboxyvinyltransferase