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.

The GTPase CpgA is implicated in the deposition of the peptidoglycan sacculus in Bacillus subtilis.

Depletion of the Bacillus subtilis GTPase CpgA produces abnormal cell shapes, nonuniform deposition of cell wall, and five- to sixfold accumulation of peptidoglycan precursors. Nevertheless, the inherent structure of the cell wall appeared mostly unchanged. The results are consistent with CpgA being involved in coordinating normal peptidoglycan deposition.

Lipid intermediates in the biosynthesis of bacterial peptidoglycan.

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.

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 beta-carboxylate of D-Asp to the epsilon-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 alpha-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.

Glycopeptide resistance vanA operons in Paenibacillus strains isolated from soil.

The sequence and gene organization of the van operons in vancomycin (MIC of >256 microg/ml)- and teicoplanin (MIC of > or =32 microg/ml)-resistant Paenibacillus thiaminolyticus PT-2B1 and Paenibacillus apiarius PA-B2B isolated from soil were determined. Both operons had regulatory (vanR and vanS), resistance (vanH, vanA, and vanX), and accessory (vanY, vanZ, and vanW) genes homologous to the corresponding genes in enterococcal vanA and vanB operons. The vanA(PT) operon in P. thiaminolyticus PT-2B1 had the same gene organization as that of vanA operons whereas vanA(PA) in P. apiarius PA-B2B resembled vanB operons due to the presence of vanW upstream from the vanHAX cluster but was closer to vanA operons in sequence. Reference P. apiarius strains NRRL B-4299 and NRRL B-4188 were found to harbor operons indistinguishable from vanA(PA) by PCR mapping, restriction fragment length polymorphism, and partial sequencing, suggesting that this operon was species specific. As in enterococci, resistance was inducible by glycopeptides and associated with the synthesis of pentadepsipeptide peptidoglycan precursors ending in D-Ala-D-Lac, as demonstrated by D,D-dipeptidase activities, high-pressure liquid chromatography, and mass spectrometry. The precursors differed from those in enterococci by the presence of diaminopimelic acid instead of lysine in the peptide chain. Altogether, the results are compatible with the notion that van operons in soil Paenibacillus strains and in enterococci have evolved from a common ancestor.

Peptidoglycan precursor pools associated with MraY and FtsW deficiencies or antibiotic treatments.

Blocking peptidoglycan synthesis in Escherichia coli with moenomycin or vancomycin led to the accumulation of UDP-MurNAc-pentapeptide and of its immediate upstream precursors, whereas with cephaloridine or penicillin G the pool of UDP-MurNAc-pentapeptide decreased. With MraY and FtsW deficiencies the decrease of UDP-MurNAc-pentapeptide was accompanied by an increase of the upstream nucleotide precursors and the appearance of UDP-MurNAc-tetrapeptide.

Fluorescence detection-based functional assay for high-throughput screening for MraY.

We have developed a novel assay specific to MraY, which catalyzes the first membrane step in the biosynthesis of bacterial cell wall peptidoglycan. This was accomplished by using UDP-MurNAc-N(epsilon)-dansylpentapeptide, a fluorescent derivative of the MraY nucleotide substrate, and a partially purified preparation of MraY solubilized from membranes of an Escherichia coli overproducing strain. Two versions of the assay were developed, one consisting of the high-pressure liquid chromatography separation of the substrate and product (dansylated lipid I) and the other, without separation and adapted to the high-throughput format, taking advantage of the different fluorescence properties of the nucleotide and lipid I in the reaction medium. The latter assay was validated with a set of natural and synthetic MraY inhibitors.

A MurG assay which utilises a synthetic analogue of lipid I.

A standard assay for the MurG enzyme using a lipid I analogue [MurNAc(N(epsilon)-dansylpentapeptide)-pyrophosphoryl (R,S)-alpha-dihydroheptaprenol] and radioactive UDP-N-acetylglucosamine was set up. A high concentration (35%) of dimethylsulfoxide was necessary for maximal activity. Separation and quantitation were accomplished by reverse-phase high performance liquid chromatography (HPLC) in isocratic conditions and on-line radioactivity detection, thereby providing a rapid and accurate assay. The kinetic parameters of the MurG reaction were determined; the reaction was shown to also catalyse the reverse reaction at a measurable rate. A lipid I analogue containing dihydroundecaprenol as the prenyl chain turned out to be a poor MurG substrate, presumably owing to aggregation.

Synthesis of the L-alanyl-L-alanine cross-bridge of Enterococcus faecalis peptidoglycan.

The enzymatic synthesis of the complete l-alanyl(1)-l-alanine(2) side chain of the peptidoglycan precursors of Enterococcus faecalis was obtained in vitro using purified enzymes. The pathway involved alanyl-tRNA synthetase and two ligases, BppA1 and BppA2, that specifically transfer alanine from Ala-tRNA to the first and second positions of the side chain, respectively. The structure of the UDP-N-acetylmuramoyl-l-Ala-gamma-d-Glu-l-Lys(N(epsilon)-l-Ala(1)-l-Ala(2))-d-Ala-d-Ala product of BppA1 and BppA2 was confirmed by mass spectrometry (MS) and MS/MS analyses. The peptidoglycan structure of the wild-type E. faecalis strain JH2-2 was determined by tandem reverse-phase high-pressure liquid chromatography-MS revealing that most muropeptides contained two l-alanyl residues in the cross-bridges and in the free N-terminal ends. Deletion of the bppA2 gene was associated with production of muropeptides containing a single alanyl residue at these positions. The relative abundance of monomers, dimers, trimers, and tetramers in the peptidoglycan of the bppA2 mutant indicated that precursors containing an incomplete side chain were efficiently used by the dd-transpeptidases in the cross-linking reaction. However, the bppA2 deletion impaired expression of intrinsic beta-lactam resistance suggesting that the low affinity penicillin-binding protein 5 did not function optimally with precursors substituted by a single alanine.

Balance between two transpeptidation mechanisms determines the expression of beta-lactam resistance in Enterococcus faecium.

The d,d-transpeptidase activity of high molecular weight penicillin-binding proteins (PBPs) is essential to maintain cell wall integrity as it catalyzes the final cross-linking step of bacterial peptidoglycan synthesis. We investigated a novel beta-lactam resistance mechanism involving by-pass of the essential PBPs by l,d-transpeptidation in Enterococcus faecium. Determination of the peptidoglycan structure by reverse phase high performance liquid chromatography coupled to mass spectrometry revealed that stepwise selection for ampicillin resistance led to the gradual replacement of the usual cross-links generated by the PBPs (d-Ala(4) --> d-Asx-Lys(3)) by cross-links resulting from l,d-transpeptidation (l-Lys(3) --> d-Asx-Lys(3)). This was associated with no modification of the level of production of the PBPs or of their affinity for beta-lactams, indicating that altered PBP activity was not required for ampicillin resistance. A beta-lactam-insensitive l,d-transpeptidase was detected in membrane preparations of the parental susceptible strain. Acquisition of resistance was not because of variation of this activity. Instead, selection led to production of a beta-lactam-insensitive d,d-carboxypeptidase that cleaved the C-terminal d-Ala residue of pentapeptide stems in vitro and caused massive accumulation of cytoplasmic precursors containing a tetrapeptide stem in vivo. The parallel dramatic increase in the proportion of l-Lys(3) --> d-Asx-Lys(3) cross-links showed that the enzyme was activating the resistance pathway by generating the substrate for the l,d-transpeptidase.

Phosphinate Inhibitors of the D-Glutamic Acid-Adding Enzyme of Peptidoglycan Biosynthesis.

We report the synthesis and initial evaluation of the first effective inhibitors of the D-glutamic acid-adding enzyme (UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase or MurD). This enzyme plays a key role in bacterial peptidoglycan biosynthesis and is therefore a target for antibiotic design. Phosphinic acid 3 is a dipeptide analog linked to uridine diphosphate by a hydrophobic spacer. It is a good inhibitor of the enzyme (IC(50) = 0.68 &mgr;M) as it closely resembles the tetrahedral intermediate that is presumed to form in the ligation reaction. Compound 4 lacks the terminal UMP group, and compound 5 lacks both the linker and UDP functionalities. These are less effective inhibitors of the enzyme with IC(50) values of 29 &mgr;M and >1 mM, respectively. Preincubation of the enzyme in the presence of inhibitor 3 and ATP does not result in irreversible inhibition or in the formation of a slowly decomplexing species, suggesting that the phosphinic acid is not phosphorylated in the active site.