A New Mechanism of Action Proposed for Ramoplanin

Abstract

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...

Complexation of peptidoglycan intermediates by the lipoglycodepsipeptide antibiotic ramoplanin: Minimal structural requirements for intermolecular complexation and fibril formation

“…Walker's verification of transglycosylase (TGase) inhibition by ramoplanin in vivo suggests that the primary mechanism of bacterial killing involves Lipid II sequestration (15). However, ramoplanin differs from transglycosylase inhibitors such as moenomycin and mersacidin in that it not only inhibits TGase activity, but also generates a metabolic block at the level of the MurG reaction, forcing a buildup of cellular pools of Lipid I (12,13).…”

“…Brotz and coworkers demonstrated that ramoplanin altered the chromatographic migration profiles of both Lipid I and Lipid II, suggesting that the two form a complex with the antibiotic, thereby raising the possibility that ramoplanin might also interfere with transglycosylation via capture of Lipid II (1). Using citronellyl-Lipid II, a soluble synthetic analogue of Lipid II, Walker and coworkers (15) subsequently confirmed that ramoplanin indeed possessed this second PG biosynthesis inhibitory activity by directly showing its inhibition of extracellular transglycosylase activity. Importantly, it was also shown that on complexation with citronellyl-Lipid II, ramoplanin underwent a ligand-induced aggregation to produce insoluble fibrils, precluding molecular characterization of the inhibitory complex.…”

Vancomycin analogues active against vanA-resistant strains inhibit bacterial transglycosylase without binding substrate

“…Lipid I analogue 9 was prepared as described in Men et al (9) and Ye et al (10) and enzymatically converted to lipid II analogue 10 by using E. coli MurG as described in Lo et al (11).…”

A crystal structure of a dimer of the antibiotic ramoplanin illustrates membrane positioning and a potential Lipid II docking interface

“…Lipid II sequestration precludes formation of the mature, fully cross-linked peptidoglycan, leading to a mechanically weakened cell wall and bacterial death from osmotic lysis (11)(12)(13)(14). Thus, like the glycopeptide antibiotics, ramoplanin inhibits cell wall biosynthesis, but the molecular underpinnings are distinct, since the binding epitopes on Lipid II that are recognized by the two types of antibiotics are largely non-overlapping (3,4,10,15,16).…”

“…Determining the stoichiometry with which Lipid II binds ramoplanin has been complicated. Early circular dichroism titrations suggested a 1:1 stoichiometry (14). However, this value was subsequently revised after analysis of the effects of ramoplanin on the conversion of heptaprenyl Lipid I to heptaprenyl Lipid II by the transglycosylase/transpeptidase PBP1b; these experiments led to an estimate of a 2:1 stoichiometry for the ramoplanin:Lipid II complex (17).…”