Inhibitors and mechanism of phospho-N-acetylmuramyl-pentapeptide translocase (Escherichia coli)

Abstract

Bacterial drug resistance is an increasingly serious problem facing public health. Consequently there is a continuing need for the development of novel antibacterial agents as therapeutic antibiotics. Bacterial peptidoglycan biosynthesis is a good target for antimicrobial agents since there is no equivalent in mammalian cells.

The first step in the membrane cycle of reactions of bacterial peptidoglycan biosynthesis is transfer of phospho-MurNAc-pentapeptide from UMP to a membrane bound carrier molecule, undecaprenyl phosphate. This step remains an unexploited potential target for antibiotics. This reaction is catalysed by the integral membrane enzyme, phospho-N-acetylmuramyl-pentapeptide translocase (translocase I). The gene coding for this enzyme in Escherichia coli, mraY, has recently been cloned. Two novel classes of antibiotics, the mureidomycins and liposidomycins, have recently been characterised as potent and specific inhibitors of translocase 1.

Translocase I (E. coli) has been overexpressed 30-fold in E. coli and has been solubilised from particulate membranes with retention of catalytic activity. A continuous fluorescence-based assay for translocase I activity has been developed based on the work of Weppner and Neuhaus (W.A. Weppner and F.C. Neuhaus 1977, J. Biol, Chem. 252, 2296-2303). Mureidomycin A is a potent slow-binding inhibitor of translocase I activity with Kj and Kj* values of 36 nM and 2 nM respectively. Liposidomycin B also appears to be a slow-binding inhibitor with a Kj* value of 143 nM.

Evidence has been obtained for a covalent enzyme-linked intermediate involved in the reaction pathway implying a ping pong type mechanism. The intermediate was labile to hydroxylamine suggesting an acyl-phosphate in the active-site.

Sequence alignments have revealed an evolutionary superfamily of phosphor-aminosugar transferase enzymes, of which translocase I is a member. Three aspartate residues conserved through this class of enzymes have been investigated by site-directed mutagenesis as possible active-site nucleophiles. The results are discussed in the context of rational drug design.

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