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-beta-1, 4-N-acetylmuramoyl-L-alanyl-gamma-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) PBP1bgamma. This derivative provides the host cell in which it is produced with a functional wall peptidoglycan. His tag (M46-N844) PBP1bgamma possesses an amino-terminal hydrophobic segment, which serves as transmembrane spanner of the native PBP. This segment is linked, via an congruent with 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 congruent with 39 000 M-1 s-1. Glu-233, of motif 1, is central to the catalysed reaction. It is proposed that the Glu-233 gamma-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

When Drug Inactivation Renders the Target Irrelevant to Antibiotic Resistance: A Case Story with Beta-Lactams

By challenging the efficiency of some of our most useful antimicrobial weapons, bacterial antibiotic resistance is becoming an increasingly worrying clinical problem. A good antibiotic is expected to exhibit a high affinity for its target and to reach it rapidly, while escaping chemical modification by inactivating enzymes and elimination by efflux mechanisms. A study of the behaviour of a beta-lactamase-overproducing mutant of Enterobacter cloacae in the presence of several penicillins and cephalosporins showed that the minimum inhibitory concentration (MIC) values for several compounds were practically independent of the sensitivity of the target penicillin binding protein (PBP), even for poor beta-lactamase substrates. This apparent paradox was explained by analysing the equation that relates the antibiotic concentration in the periplasm to that in the external medium. Indeed, under conditions that are encountered frequently in clinical isolates, the factor characterizing the PBP sensitivity became negligible. The conclusions can be extended to all antibiotics that are sensitive to enzymatic inactivation and efflux mechanisms and must overcome permeability barriers. It would be a grave mistake to neglect these considerations in the design of future antibacterial chemotherapeutic agents