IntroductionPeptidoglycan is a crosslinked carbohydrate polymer that surrounds bacterial cells and prevents them from rupturing under high internal osmotic pressures. Because peptidoglycan is essential for survival and has no eukaryotic counterpart, peptidoglycan biosynthesis (Fig. 1) is the target of a large number of clinically used antibiotics, including the β-lactams, cephalosporins, and glycopeptide antibiotics.1The emergence of resistance to all these classes of antibiotics represents a significant threat to public health and has stimulated efforts to develop structurally novel antibacterial agents that inhibit the peptidoglycan biosynthetic pathway. One molecule that has received considerable attention in recent years is ramoplanin (Fig. 2,1), a lipoglycodepsipeptide antibiotic discovered in the 1980s in a screen for peptidoglycan synthesis inhibitors.2,3Ramoplanin has good activity against a wide range of Gram-positive organisms and is regarded as a promising candidate for the treatment of many Gram-positive infections.4It is currently in late stage clinical trials for two different indications.5,6Due to hydrolytic instability and other issues, however, neither of these indications involves systemic administration of ramoplanin, and the full potential of this compound has yet to be realized.4A better understanding of the mechanism of action of ramoplanin may enable the development of derivatives to treat systemic infections.Peptidoglycan biosynthesis.Structure of ramoplanin (1), enduracidin (2) and the ramoplanin aglycon (3).A mechanism of action for ramoplanin was first proposed in 1990 by Somner and Reynolds, who showed, using a cell-free, particulate membrane assay, that the antibiotic blocks the MurG-catalyzed conversion of Lipid I to Lipid II (Fig. 1) on the biosynthetic pathway to peptidoglycan.7Although no direct evidence for an interaction with Lipid I was presented, these authors suggested that ramoplanin kills bacterial cells by binding to this substrate, rendering it inaccessible to MurG.7A decade later, also using a cell-free, particulate membrane system, we showed that ramoplanin inhibits the transgly-cosylase-catalyzed coupling of Lipid II molecules to form the carbohydrate chains of peptidoglycan.8We established that ramoplanin binds to synthetic Lipid II analogues, and so we proposed that ramoplanin acts primarily by binding to Lipid II and inhibiting the transglycosylation step of peptidoglycan biosynthesis.8,9Our hypothesis that the primary mechanism of action of ramoplanin involves binding to Lipid II and inhibition of transglycosylation rather than binding to Lipid I and inhibition of MurG rested largely on the fact that Lipid II is translocated to the external surface of the bacterial membrane as soon as it is produced whereas Lipid I remains on the internal surface of the membrane.10Ramoplanin is a large and highly water-soluble molecule, and in the absence of a dedicated transport mechanism, it seemed improbable that it could diffuse readily through bacterial membranes to reach an intracellular target. In fact, Somner and Reynolds made this point in their mechanistic papers on ramoplanin, but when they did their studies it was not known that Lipid I and MurG are intracellular.7,10Comparative information on how well ramoplanin and various analogues inhibit MurG and the bacterial trans-glycosylases could provide more insight into the mechanism of action of the molecule. We have developed synthetic routes to Lipid I and Lipid II substrates and have developed assays to studyE. coliMurG andE. coliPBP1b, the major bacterial transglycosylase in this organism.9,11,12These tools enable us to carry out the studies required to assess the importance of the different proposed targets of ramoplanin and structurally related compounds. Below we report a comparative analysis of the inhibition kinetics of ramoplanin, the ramoplanin aglycon (Fig. 2,3), and the related antibiotic enduracidin (Fig. 2,2) with respect to MurG and PBP1b. We also present quantitative information on the binding affinities of ramoplanin for Lipid I and Lipid II. These studies support the hypothesis that the primary mechanism of action of ramoplanin involves binding to Lipid II and inhibiting the transglycosylation step of peptidoglycan biosynthesis. They also indicate that enduracidin operates by the same mechanism as ramoplanin.Structures of fluorescein-labeled peptidoglycan precursors.