Ramoplanin (Figure 1) is a cyclic glycolipodepsipeptide antibiotic that kills gram positive bacteria by inhibiting cell wall biosynthesis. Ramoplanin was shown to block the conversion of Lipid I to Lipid II, 1 a reaction that is catalyzed by the intracellular GlcNAc transferase, MurG (Scheme 1). It was proposed that ramoplanin inhibits MurG by complexing Lipid I, which prevents it from being utilized as a substrate. Below we show that ramoplanin also inhibits the polymerization of Lipid II; therefore, we propose that another mechanism by which ramoplanin can kill bacterial cells is through inhibition of the transglycosylation step of peptidoglycan synthesis. Using a synthetic analogue of Lipid II, we present evidence that enzyme inhibition by ramoplanin involves substrate binding. Ramoplanin undergoes a conformational change upon substrate binding, and the resulting complexes self-associate to form fibrils. The significance of fibril formation is discussed.The mechanism of action of ramoplanin has been investigated in permeabilized bacterial cells and membrane preparations by following the incorporation of radiolabel from a precursor into various intermediates along the pathway to peptidoglycan. 1-3 A limitation of these assays is that if one enzymatic step is blocked, then no information can be obtained about subsequent steps. Thus, because ramoplanin prevents the formation of Lipid II, it is not possible to determine whether it also inhibits the polymerization of Lipid II. We reinvestigated the ability of ramoplanin to block Lipid II polymerization using a modified membrane assay 4 in which the transglycosylases are selectively inhibited to permit the buildup of radiolabeled Lipid II. Following removal of the inhibitor, peptidoglycan synthesis commences. The effect of ramoplanin on Lipid II polymerization was evaluated by monitoring the amount of radioactive peptidoglycan formed in the presence of increasing concentrations of ramoplanin. Ramoplanin blocks the polymerization of Lipid II and thus is an inhibitor of the transglycosylation step of peptidoglycan synthesis (Figure 2).Ramoplanin was proposed to act by complexing substrates required for peptidoglycan synthesis. 1 Unfortunately, difficulties in isolating Lipid intermediates from bacterial cells have hindered studies of their interactions with ramoplanin. 5,6 Moreover, the natural Lipid intermediates contain a 55 carbon polyprenol chain that renders them insoluble in water, and thus difficult to use in biophysical studies of complex formation. We recently developed a synthetic route to a soluble Lipid I analogue (1) to use in studying MurG, 7 the GlcNAc transferase that converts Lipid I to Lipid II. Using purified MurG, we have now made the corresponding Lipid II analogue 2 from 1, as shown (Scheme 2). 8,9 Compound 2 is identical to natural Lipid II except that the 55 carbon chain has been replaced with a 10 carbon unit so that the compound is freely water soluble. 10 The ability of ramoplanin to interact with 2 was investigated by NMR (Figure 3). Ti...