Jean van Heijenoort

The catalytic, glycosyl transferase and acyl transferase modules of the cell wall peptidoglycan‐polymerizing penicillin‐binding protein 1b of Escherichia coli

The penicillin‐binding protein (PBP) 1b of Escherichia coli catalyses the assembly of lipid‐transported N‐acetyl glucosaminyl‐β‐1,4‐N‐acetylmuramoyl‐l‐alanyl‐γ‐d‐glutamyl‐(l)‐meso‐diaminopimelyl‐(l)‐d‐alanyl‐d‐alanine disaccharide pentapeptide units into polymeric peptidoglycan. These units are phosphodiester linked, at C1 of muramic acid, to a C55 undecaprenyl carrier. PBP1b has been purified in the form of His tag (M46‐N844) PBP1bγ. This derivative provides the host cell in which it is produced with a functional wall peptidoglycan. His tag (M46‐N844) PBP1bγ possesses an amino‐terminal hydrophobic segment, which serves as transmembrane spanner of the native PBP. This segment is linked, via an ≅ 100‐amino‐acid insert, to a D198‐G435 glycosyl transferase module that possesses the five motifs characteristic of the PBPs of class A. In in vitro assays, the glycosyl transferase of the PBP catalyses the synthesis of linear glycan chains from the lipid carrier with an efficiency of ≅ 39 000 M−1 s−1. Glu‐233, of motif 1, is central to the catalysed reaction. It is proposed that the Glu‐233 γ‐COOH donates its proton to the oxygen atom of the scissile phosphoester bond of the lipid carrier, leading to the formation of an oxocarbonium cation, which then undergoes attack by the 4‐OH group of a nucleophile N‐acetylglucosamine. Asp‐234 of motif 1 or Glu‐290 of motif 3 could be involved in the stabilization of the oxocarbonium cation and the activation of the 4‐OH group of the N‐acetylglucosamine. In turn, Tyr‐310 of motif 4 is an important component of the amino acid sequence‐folding information. The glycosyl transferase module of PBP1b, the lysozymes and the lytic transglycosylase Slt70 have much the same catalytic machinery. They might be members of the same superfamily. The glycosyl transferase module is linked, via a short junction site, to the amino end of a Q447‐N844 acyl transferase module, which possesses the catalytic centre‐defining motifs of the penicilloyl serine transferases superfamily. In in vitro assays with the lipid precursor and in the presence of penicillin at concentrations sufficient to derivatize the active‐site serine 510 of the acyl transferase, the rate of glycan chain synthesis is unmodified, showing that the functioning of the glycosyl transferase is acyl transferase independent. In the absence of penicillin, the products of the Ser‐510‐assisted double‐proton shuttle are glycan strands substituted by cross‐linked tetrapeptide–pentapeptide and tetrapeptide–tetrapeptide dimers and uncross‐linked pentapeptide and tetrapeptide monomers. The acyl transferase of the PBP also catalyses aminolysis and hydrolysis of properly structured thiolesters, but it lacks activity on d‐alanyl‐d‐alanine‐terminated peptides. This substrate specificity suggests that carbonyl donor activity requires the attachment of the pentapeptides to the glycan chains made by the glycosyl transferase, and it implies that one and the same PBP molecule catalyses transglycosylation and peptide cross‐linking in a sequential manner. Attempts to produce truncated forms of the PBP lead to the conclusion that the multimodular polypeptide chain behaves as an integrated folding entity during PBP1b biogenesis.

Lipid Intermediates in the Biosynthesis of Bacterial Peptidoglycan

SUMMARY This review is an attempt to bring together and critically evaluate the now-abundant but dispersed data concerning the lipid intermediates of the biosynthesis of bacterial peptidoglycan. Lipid I, lipid II, and their modified forms play a key role not only as the specific link between the intracellular synthesis of the peptidoglycan monomer unit and the extracytoplasmic polymerization reactions but also in the attachment of proteins to the bacterial cell wall and in the mechanisms of action of antibiotics with which they form specific complexes. The survey deals first with their detection, purification, structure, and preparation by chemical and enzymatic methods. The recent important advances in the study of transferases MraY and MurG, responsible for the formation of lipids I and II, are reported. Various modifications undergone by lipids I and II are described, especially those occurring in gram-positive organisms. The following section concerns the cellular location of the lipid intermediates and the translocation of lipid II across the cytoplasmic membrane. The great efforts made since 2000 in the study of the glycosyltransferases catalyzing the glycan chain formation with lipid II or analogues are analyzed in detail. Finally, examples of antibiotics forming complexes with the lipid intermediates are presented.Summary: This review is an attempt to bring together and critically evaluate the now-abundant but dispersed data concerning the lipid intermediates of the biosynthesis of bacterial peptidoglycan. Lipid I, lipid II, and their modified forms play a key role not only as the specific link between the intracellular synthesis of the peptidoglycan monomer unit and the extracytoplasmic polymerization reactions but also in the attachment of proteins to the bacterial cell wall and in the mechanisms of action of antibiotics with which they form specific complexes. The survey deals first with their detection, purification, structure, and preparation by chemical and enzymatic methods. The recent important advances in the study of transferases MraY and MurG, responsible for the formation of lipids I and II, are reported. Various modifications undergone by lipids I and II are described, especially those occurring in gram-positive organisms. The following section concerns the cellular location of the lipid intermediates and the translocation of lipid II across the cytoplasmic membrane. The great efforts made since 2000 in the study of the glycosyltransferases catalyzing the glycan chain formation with lipid II or analogues are analyzed in detail. Finally, examples of antibiotics forming complexes with the lipid intermediates are presented.

Topological analysis of the MraY protein catalysing the first membrane step of peptidoglycan synthesis

The two‐dimensional membrane topology of the Escherichia coli and Staphylococcus aureus MraY transferases, which catalyse the formation of the first lipid intermediate of peptidoglycan synthesis, was established using the β‐lactamase fusion system. All 28 constructed mraY–blaM fusions produced hybrid proteins. Analysis of the ampicillin resistance of the strains with hybrids led to a common topological model possessing 10 transmembrane segments, five cytoplasmic domains and six periplasmic domains including the N‐ and C‐terminal ends. The agreement between the topologies of E. coli and S. aureus, their agreement to a fair extent with predicted models and a number of features arising from the comparative analysis of 25 orthologue sequences strongly suggested the validity of the model for all eubacterial MraYs. The primary structure of the 10 transmembrane segments diverged among orthologues, but they retained their hydrophobicity, number and size. The similarity of the sequences and distribution of the five cytoplasmic domains in both models, as well as their conservation among the MraY orthologues, clearly suggested their possible involvement in substrate recognition and in the catalytic process. Complementation tests showed that only fusions with untruncated mraY restored growth. It was noteworthy that S. aureus MraY was functional in E. coli. An increased MraY transferase activity was observed only with the untruncated hybrids from both organisms.

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)

Peptidoglycan Hydrolases of Escherichia coli

The review summarizes the abundant information on the 35 identified peptidoglycan (PG) hydrolases of Escherichia coli classified into 12 distinct families, including mainly glycosidases, peptidases, and amidases. An attempt is also made to critically assess their functions in PG maturation, turnover, elongation, septation, and recycling as well as in cell autolysis. There is at least one hydrolytic activity for each bond linking PG components, and most hydrolase genes were identified. Few hydrolases appear to be individually essential. The crystal structures and reaction mechanisms of certain hydrolases having defined functions were investigated. However, our knowledge of the biochemical properties of most hydrolases still remains fragmentary, and that of their cellular functions remains elusive. Owing to redundancy, PG hydrolases far outnumber the enzymes of PG biosynthesis. The presence of the two sets of enzymes acting on the PG bonds raises the question of their functional correlations. It is difficult to understand why E. coli keeps such a large set of PG hydrolases. The subtle differences in substrate specificities between the isoenzymes of each family certainly reflect a variety of as-yet-unidentified physiological functions. Their study will be a far more difficult challenge than that of the steps of the PG biosynthesis pathway.SUMMARY The review summarizes the abundant information on the 35 identified peptidoglycan (PG) hydrolases of Escherichia coli classified into 12 distinct families, including mainly glycosidases, peptidases, and amidases. An attempt is also made to critically assess their functions in PG maturation, turnover, elongation, septation, and recycling as well as in cell autolysis. There is at least one hydrolytic activity for each bond linking PG components, and most hydrolase genes were identified. Few hydrolases appear to be individually essential. The crystal structures and reaction mechanisms of certain hydrolases having defined functions were investigated. However, our knowledge of the biochemical properties of most hydrolases still remains fragmentary, and that of their cellular functions remains elusive. Owing to redundancy, PG hydrolases far outnumber the enzymes of PG biosynthesis. The presence of the two sets of enzymes acting on the PG bonds raises the question of their functional correlations. It is difficult to understand why E. coli keeps such a large set of PG hydrolases. The subtle differences in substrate specificities between the isoenzymes of each family certainly reflect a variety of as-yet-unidentified physiological functions. Their study will be a far more difficult challenge than that of the steps of the PG biosynthesis pathway.

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

Chapter 3 Biosynthesis of the bacterial peptidoglycan unit

Publisher Summary This chapter discusses the biosynthesis of the bacterial peptidoglycan unit. The biosynthesis of bacterial peptidoglycan is a complex two stage process. The first stage is concerned with the formation of the disaccharide peptide monomer unit, and the second the polymerization reactions accompanied by the insertion of the newly made peptidoglycan material into the cell wall. The assembly of the peptidoglycan unit proceeds by a series of cytoplasmic and membrane reactions. This implies a passage through the hydrophobic environment of the membrane. Lipid intermediates are involved in this process. The various steps of the pathway have been identified in one bacteria or another, and a general scheme established that is valid for both Gram-positive and negative eubacteria. The elucidation of the pathway leading to the complete peptidoglycan unit was established by isolating and characterizing the muramic acid containing precursors, and by developing a specific in vitro assay for the enzymatic activity catalyzing each step. Assays involving more than one step have also been developed.

Moenomycin A: The role of the methyl group in the moenuronamide unit and a general discussion of structure-activity relationships

Abstract Two disaccharide analogues 1b and 17a of moenomycin A have been synthesized and their antibiotic and transglycosylase-inhibiting properties have been determined. The results permit for the first time to arrive at a general view of the structural requirements in this class of compounds necessary to elicit antibiotic activity.

Aslfm, the D-aspartate ligase responsible for the addition of D-aspartic acid onto the peptidoglycan precursor of Enterococcus faecium.

d-Aspartate ligase has remained the last unidentified peptide bond-forming enzyme in the peptidoglycan assembly pathway of Gram-positive bacteria. Here we show that a two-gene cluster of Enterococcus faecium encodes aspartate racemase (Racfm) and ligase (Aslfm) for incorporation of d-Asp into the side chain of the peptidoglycan precursor. Aslfm was identified as a new member of the ATP-grasp protein superfamily, which includes a diverse set of enzymes catalyzing ATP-dependent carboxylate-amine ligation reactions. Aslfm specifically ligated the β-carboxylate of d-Asp to the ϵ-amino group of l-Lys in the nucleotide precursor UDP-N-acetylmuramyl-pentapeptide. d-iso-Asparagine was not a substrate of Aslfm, indicating that the presence of this amino acid in the peptidoglycan of E. faecium results from amidation of the α-carboxyl of d-Asp after its addition to the precursor. Heterospecific expression of the genes encoding Racfm and Aslfm in Enterococcus faecalis led to production of stem peptides substituted by d-Asp instead of l-Ala2, providing evidence for the in vivo specificity and function of these enzymes. Strikingly, sequencing of the cross-bridges revealed that substitution of l-Ala2 by d-Asp is tolerated by the d,d-transpeptidase activity of the penicillin-binding proteins both in the acceptor and in the donor substrates. The Aslfm ligase appears as an attractive target for the development of narrow spectrum antibiotics active against multiresistant E. faecium.

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.