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 coil catalyses the a ssembly of lipid-transported N-acetyl glucosaminyl-beta-1,4-N-acetylmuramoy l-L-alanyl-gamma-D-glutamyl-(L)-meso-diaminopimelyl-(L)-D-alanyl-alanine di saccharide pentapeptide units into polymeric peptidoglycan. These units are phosphodiester linked, at C1 of muramic acid, to a C55 undecaprenyl carrie r. PBP1b has been purified in the form of His tag (M46-N844) PBP1b gamma. T his derivative provides the host cell in which it is produced with a functi onal wall peptidoglycan. His tag (M46-N844) PBP1b gamma possesses an amino- terminal hydrophobic segment, which serves as transmembrane spanner of the native PBP. This segment is linked, via an congruent to 100-amino-acid inse rt, to a D198-G435 glycosyl transferase module that possesses the five moti fs 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 to 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 scissi le phosphoester bond of the lipid carrier, leading to the formation of an o xocarbonium cation, which then undergoes attack by the 4-OH group of a nucl eophile 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 activa tion of the 4-OH group of the N-acetylglucosamine. In turn, Tyr-310 of moti f 4 is an important component of the amino acid sequence-folding informatio n. The glycosyl transferase module of PBP1b, the lysozymes and the lytic tr ansglycosylase 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 transferas e module, which possesses the catalytic centre-defining motifs of the penic illoyl serine transferases superfamily. In in vitro assays with the lipid p recursor 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 g lycosyl transferase is acyl transferase independent. In the absence of peni cillin, the products of the Ser-510-assisted double-proton shuttle are glyc an strands substituted by cross-linked tetrapeptide-pentapeptide and tetrap eptide-tetrapeptide dimers and uncross-linked pentapeptide and tetrapeptide monomers. The acyl transferase of the PBP also catalyses aminolysis and hy drolysis of properly structured thiolesters, but it lacks activity on D-ala nyl-D-alanine-terminated peptides. This substrate specificity suggests that carbonyl donor activity requires the attachment of the pentapeptides to th e glycan chains made by the glycosyl transferase, and it implies that one a nd the same PBP molecule catalyses transglycosylation and peptide cross-lin king 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.