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

Abstract

Bacterial transglycosylases are enzymes that couple the disaccharide subunits of peptidoglycan to form long carbohydrate chains. These enzymes are the target of the pentasaccharide antibiotic moenomycin as well as the proposed target of certain glycopeptides that overcome vancomycin resistance. Because bacterial transglycosylases are difficult enzymes to study, it has not previously been possible to evaluate how moenomycin inhibits them or to determine whether glycopeptide analogues directly target them. We have identified transglycosylase assay conditions that enable kinetic analysis of inhibitors and have examined the inhibition of Escherichia coli penicillin-binding protein 1b (PBP1b) by moenomycin as well as by various glycopeptides. We report that chlorobiphenyl vancomycin analogues that are incapable of binding substrates nevertheless inhibit E. coli PBP1b, which shows that these compounds interact directly with the enzyme. These findings support the hypothesis that chlorobiphenyl vancomycin derivatives overcome vanA resistance by targeting bacterial transglycosylases. We have also found that moenomycin is not competitive with respect to the lipid II substrate of PBP1b, as has long been believed. With the development of suitable methods to evaluate bacterial transglycosylases, it is now possible to probe the mechanism of action of some potentially very important antibiotics.

Crystal structure of a peptidoglycan glycosyltransferase suggests a model for processive glycan chain synthesis

“…The kinetic parameters for the WT enzyme are k cat ϭ 3.5 min Ϫ1 and k cat /K m ϭ 6 ϫ 10 5 M Ϫ1 ⅐min Ϫ1 . This truncated PGT domain thus has comparable activity to the most efficient PGT studied to date, full length E. coli PBP1B (16,21,22), which contains the transmembrane helix and the TP domain. Activity was not detectable for either the E83 or the D84 mutant (E83A, E83Q, D84A, and D84N) under our standard assay conditions, which can only detect turnover greater than 0.04 min Ϫ1 ; however, turnover was observed for D84N, but not E83Q, at high enzyme concentrations and extended reaction times.…”

“…Kinetic parameters were obtained for the WT PGT domain by measuring reaction rates in assay buffer (50 mM Hepes, pH 7.5/10 mM CaCl 2 /20% DMSO) at varying concentrations (0.5-20 M) of ([ 14 C]-GlcNAc)-heptaprenyl Lipid II (LII*, 288 Ci/ mol) (1 Ci ϭ 37 GBq), as described by Chen et al (21). Standard reactions for the mutants (5 l each) were carried out in nonstick PCR tubes containing 60 nM enzyme, assay buffer, and 4 M LII*.…”

Peripheral modifications of [Ψ[CH 2 NH]Tpg 4 ]vancomycin with added synergistic mechanisms of action provide durable and potent antibiotics

“…Vancomycin is also thought to inhibit the preceding step in the cell wall biosynthesis, the transglycosylasecatalyzed incorporation of lipid intermediate II into the polysaccharide backbone of the bacterial cell wall. In the case of vancomycin, this inhibition also requires D-Ala-D-Ala binding (30)(31)(32)(33). However, it is not yet clear whether this occurs through direct binding of the vancomycin disaccharide to the enzyme active site, because cell wall binding contributes to its localization, or whether this occurs by indirect enzyme inhibition.…”

The molecular biology of moenomycins: towards novel antibiotics based on inhibition of bacterial peptidoglycan glycosyltransferases

“…In vitro, inhibitory activity of a delipidated derivative of MmA (4, Figure 1) decreases 100-fold, yet its potency is still comparable to that of clinically useful glycopeptides. Unfortunately, in vivo antibacterial activity of the delipidated MmA is completely abolished (Chen et al, 2003). Likewise, an MmA analog containing C 10 lipid (5; Figure 1) is shown to efficiently inhibit PGTs in vitro, although its antibacterial activity in vivo is negligible (Adachi et al, 2006).…”

“…Since natural products produced by microorganisms and plants evolved to function in environments very different from that of the human body, their structures often need to be modified to improve in vivo activity and/or pharmacology. Although chemical synthesis has been widely used to achieve such structural improvements (Welzel, 2005), biology-based approaches to manipulate complicated natural product chemistry are now competitive with these chemical methodologies in many cases (Chen et al, 2003). In these approaches, genes or enzymes for natural product biosynthesis are 'mixed' to generate novel metabolic pathways, ultimately leading to a library of closely related compounds.…”