Yveline van Heijenoort

In vitro peptidoglycan polymerization catalysed by penicillin binding protein 1b of Escherichia coli K-12

The discovery of penicillin binding proteins (PBP) in bacteria [ 1,2] and the isolation of mutants defective in these proteins [3,4] have provided a new approach to the study of the P-lactam-sensitive enzymes involved in the biosynthesis of peptidoglycan and of their correlation with cell elongation and cell division. In Escherichia coli, 7 PBPs have been described and their possible physiological roles speculated [3-61. In particular, it was suggested that PBP-lb is directly involved in the polymerization steps of the biosynthesis of peptidoglycan [4,5,7,8]. This polymerization is known [9,10] to proceed at the expense of the lipid intermediate N-acetylglucosaminyl-N-acetylmuramyl(pentapeptide)-pyrophosphoryl-undecaprenol by formation of the linear glycan strands (transglycosylation step) and crosslinking of the peptide subunits (transpeptidation step). The data presented here clearly substantiate the fact that PBPlb can catalyse polymerization from the purified specifically radiolabelled lipid intermediate. This was established in two different ways: (1)

Polymerization by transglycosylation in the biosynthesis of the peptidoglycan of Escherichia coli K 12 and its inhibition by antibiotics

The biosynthesis of bacterial cell wall peptidoglycan is a complex process involving cytoplasmic and membrane steps [l] . N-Acetylglucosaminyl-N-acetylmuramyl-(pentapeptide)-pyrophosphoryl-undecaprenol is the last membrane precursor prior to polymerization which proceeds by transglycosylation (formation of the linear glycan strands) and transpeptidation (crosslinking of the peptide subunits) [l] . This membrane intermediate has been utilized in cellfree systems for the formation of peptidoglycan [2-l] , but the difficulty in conveniently isolating it in adequate amounts has greatly limited the direct investigation of the polymerizing enzymes. Most commonly, the in vitro synthesis of peptidoglycan is carried out by incubating the cytoplasmic precursors, UDP-iV-acetylmuramyl-pentapeptide and UDP-Nacetylglucosamine, with appropriate particulate preparations, crude cell walls or treated cells [3-lo] (see [l] for ref. before 1972). Under these conditions only the over-all course of the different membrane reactions is considered. However, a study of each membrane step in itself is essential for the understanding of the mode of action of certain antibiotics, for the development of the genetic analysis of these reactions and for the determination of the mechanisms involved in their regulation. Recently, the transpeptidation step, uncoupled from the other membrane reactions, has been successfully studied with artificial systems of donor and acceptor peptides [ 1 ,111. As far as we are aware, the transglycosylation step has not yet been directly investigated to any extent. The present paper describes a convenient in vitro

Moenomycin A: A structural revision and new structure-activity reactions

Abstract A detailed FAB MS analysis combined with NMR and chemical results requires the structure of moenomycin A to be revised from 1a to 1b. New bio-chemical results seem to support the assumption that in the region F-G-H of lb the structural requirements for antibiotic activity are rather strict.

Biosynthesis of the peptidoglycan of Escherichia coli K-12: Properties of the in vitro polymerization by transglycosylation

In the biosynthesis of bacterial peptidoglycan polymerization proceeds at the expense of the lipid intermediate ~-acetyl~ucosam~yl-~-acetylmuramyl(pentapeptide).pyrophospho~l-undecaprenol by the formation of the linear glycan strands (transglycosylation step) and the cross-linking of the peptide subunits ~tr~speptidation step) [ 1 ]. A convenient in vitro assay specific for the tr~sglycosylation step was described in ]2]. The in vitro formation of polymerized uncrosslinked peptidoglycan material from the lipid ~termediate was catalysed by particulate fractions from Escherichia coii in the presence of sodium deoxycholate and penicillin G. Here we report general properties of this in vitro polymerization reaction as well as a more complete investigation of the effect of different antibiotics. The importance of this reaction is stressed by the fact that purified penicillin binding protein lb alone can catalyse a similar reaction [3,4].

A structurally and biogenetically interesting moenomycin antibiotic

Isolation and structure elucidation of a new moenomycin antibiotic (A12, 1a) is reported that differs from moenomycin A by lack of the branching methyl group and by the configuration at C-4 of unit F. The smallest antibiotically active degradation product of 1a is the trisaccharide derivative 3a. This observation is in contrast to structure activity relations in the moenomycin A series where it was found that disaccharide 4b is fully active. An explanation is offered for this difference.

Moenomycin a: Minimum structural requirements for biological activity

Abstract A stepwise degradation of the oligosaccharide part of moenomycin A (2) was performed. The degradation products were assayed for antibiotic activity both in vivo and in an E.coli cell-free system. Units E, F, G, H, and I haue been found to be essential for full biological ( in vitro ) activity. It is suggested that 2 is a competitive inhibitor of the peptidoglycan polymerase.

Structures of some moenomycin antibiotics-inhibitors of peptidoglycan biosynthesis

Abstract Isolation, structural assignment, and antibiotic efficiency of the new moenomycin antibiotics C3 (1b) and C4 (1c) is decribed. The previously published structure of pholipomycin (1d) is modified.

Moenomycin A: reactions at the lipid part. New structure-activity relations

Abstract Methods for the removal and the oxidation of the lipid moiety of the antibiotic moenomycin A have been studied. Combined with biochemical studies, the results demonstrate that antibiotic activity is closely related with the integrity of the lipid unit.

The first moenomycin antibiotic without the methyl-branched uronic acid constituent.- Unexpected structure activity relations

Abstract Isolation and structure elucidation of a new moenomycin antibiotic (C1, 1e) that lacks the branching methyl group in the 4-position of unit F are reported. The smallest antibiotically active degradation product of 1e is the trisaccharide derivative 3. This observation is in contrast to structure activity relations in the moenomycin A series where it was found that disaccharide 4a is fully active.

Introduction of a terminal hydroxy group into the lipid part of a moenomycin-type transglycosylase inhibitor suppresses antibiotic activity

Abstract On reaction with singlet oxygen 1 provided a mixture of 2 and 3 which could not be separated. In-vivo , 2/3 were antibiotically of low activity whereas in the in-vitro assays at least one of the compounds inhibited the transglycosylase effectively.