Monitoring UDP-MurNAc (UM)-Stem Peptide Intermediates from the Cellular and Recombinant <i>Acinetobacter baumannii</i> MurCDEF Pathway

Increasing multidrug resistance in nosocomial, opportunistic bacterial pathogens motivates a need to exploit metabolic pathways of therapeutic value. Here, we describe a one-pot reconstitution of the MurCDEF pathway from <i>Acinetobacter baumannii</i>, made possible by the biosynthesis and quantification of UDP-MurNAc (UM) and UM-stem peptide derivatives prepared in isotopically "heavy" and "light" isotopomer forms. The successive activity of each Mur ligase in one-pot is quantified by C18 liquid chromatography (LC). We show that the kinetics of sequential extension of the peptide stem is readily modulated by adjusting the relative Mur ligase concentrations or by adding effectors to the assay. We find that MurF generally pulls this pathway to mature the UM-pentapeptide product from UM, with other intermediates accumulating to variable degrees. Determination of the absolute concentrations of UM and UM-stem peptide intermediates in <i>A. baumannii</i> cells reveals, like the one-pot assay, a high relative UM-pentapeptide product, the concentration of which is decreased significantly by the antibiotic <i>D</i>-cycloserine. These in vitro and cellular assays collectively illustrate the promise of this toolkit to discover small molecules that target a specific Mur ligase or uncover novel aspects of peptidoglycan biosynthesis and recycling.