A peptide inhibitor of MurA UDP-N-acetylglucosamine enolpyruvyl transferase: The first committed step in peptidoglycan biosynthesis Jose ´ Molina-Lo ´ pez a,1 , Franc ¸ois Sanschagrin b , Roger C. Levesque b, * a Departamento de Salud Pu ´ blica, Facultad de Medicina, Universidad Nacional Auto ´noma de Me ´xico, Mexico b Centre de Recherche sur la Fonction, Structure et Inge ´nierie des Prote ´ines, Pav. C.-E. Marchand, Faculte ´ de Me ´dicine, Universite ´ Laval, Sainte-Foy, Que ´bec, Canada G1K 7P4 1. Introduction Peptidoglycan is an essential cell wall component of most bacteria and their biosynthetic pathway is a complex of two- stage process [18]. The first stage occurs in the cytoplasm, and consists in the formation of the monomeric building block N- acetylglucosamine-N-acetylmuramyl pentapeptide. This first committed step in this pathway is the transfer of an enolpyruvate residue from phosphoenolpyruvate to position 3 of UDP-N-acetylglucosamine [5]. This reaction is catalyzed by MurA enzyme (Fig. 1). This is followed by a MurB-catalyzed reduction of the enolpyruvate moiety to D-lactate, yielding UDP-N-acetylmuramate. A series of ATP-dependent amino acid ligases, MurC to MurF catalyze the stepwise addition of amino acids to the pentapeptide side-chain to form UDP-N- acetylmuramyl pentapeptide. MurA to MurF cascade is ubiquitous to both Gram-positive and Gram-negative bacteria, and has no homologue in mammalian cells [19]. The inhibition of one of MurA through MurF enzymes leads to loss of cell shape and integrity followed by bacterial cell lysis and death [8,14]. However, enzymes implicated in this path- way are not inhibited by known antibacterial agents, except for MurA, inhibited by fosfomycin [15,25] in which the active ingredient of Monurol forms a covalent adduct with a cysteine residue, Cys 115 (numbering according to MurA from Escherichia coli). Cys 115 is located in a solvent exposed loop that undergoes peptides 27 (2006) 3115–3121 article info Article history: Received 22 June 2006 Received in revised form 22 August 2006 Accepted 23 August 2006 Published on line 9 October 2006 Keywords: MurA Phage display UDP-N-acetylglucosamine Peptide inhibitors abstract 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 200 mM 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. # 2006 Elsevier Inc. All rights reserved. * Corresponding author. Tel.: +1 418 656 3070; fax: +1 418 656 7176. E-mail addresses: joseml@servidor.unam.mx (J. Molina-Lo ´ pez), fsanscha@rsvs.ulaval.ca (F. Sanschagrin), rclevesq@rsvs.ulaval.ca (R.C. Levesque). 1 Permanent address: Av. Universidad No. 3000, Ciudad Universitaria, Colonia Copilco Universidad, Mexico D.F., C.P. 04510, Mexico. available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/peptides 0196-9781/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2006.08.023