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

Abstract

SummaryThe penicillin-binding protein (PBP) 1b of Escherichia coli catalyses the assembly of lipid-transported Nacetyl glucosaminyl-b-1,4 -N-acetylmuramoyl-L-alanyl-g-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 puri®ed in the form of His tag (M46-N844) PBP1bg. This derivative provides the host cell in which it is produced with a functional wall peptidoglycan. His tag (M46-N844) PBP1bg possesses an amino-terminal hydrophobic segment, which serves as transmembrane spanner of the native PBP. This segment is linked, via an h 100-amino-acid insert, to a D198-G435 glycosyl transferase module that possesses the ®ve 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 ef®ciency of h 39 000 M À1 s À1 . Glu-233, of motif 1, is central to the catalysed reaction. It is proposed that the Glu-233 g-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-de®ning motifs of the penicilloyl serine transferases superfamily. In in vitro assays with the lipid precursor and in the presence of penicillin at concentrations suf®cient to derivatize the active-site serine 510 of the acyl transferase, the rate of glycan chain synthesis is unmodi®ed, 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 speci®city 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. Att...

Crystal structure of a peptidoglycan glycosyltransferase suggests a model for processive glycan chain synthesis

“…These results suggest that E83, and to a lesser extent D84, play important roles in the catalytic mechanism. Consistent with our findings, Terrak et al (16) have identified the invariant glutamate residue in conserved sequence motif 1 as critical for catalysis by the PGT domain of E. coli PBP1B.…”

“…The kinetic parameters for the WT enzyme are k cat ϭ 3.5 min Ϫ1 and k cat /K m ϭ 6 ϫ 10 5 M Ϫ1 ⅐min Ϫ1 . This truncated PGT domain thus has comparable activity to the most efficient PGT studied to date, full length E. coli PBP1B (16,21,22), which contains the transmembrane helix and the TP domain. Activity was not detectable for either the E83 or the D84 mutant (E83A, E83Q, D84A, and D84N) under our standard assay conditions, which can only detect turnover greater than 0.04 min Ϫ1 ; however, turnover was observed for D84N, but not E83Q, at high enzyme concentrations and extended reaction times.…”

Characterization of derivatives of the high-molecular-mass penicillin-binding protein (PBP) 1 of Mycobacterium leprae

“…in the putative C-terminal extension of PBP1b of E. coli, resulted in a protein devoid of penicillinbinding activity [24]. Through construction of a series of deletion mutants of high-molecular-mass PBPs, such as PBP1b [11] and PBP3 [10] of E.coli, PBP2h of Staphylococcus aureus [25] and PBP5 of Enterococcus hirae [26], it has been postulated that acquisition of a conformation of the PB module that is competent to bind benzylpenicillin requires the presence of the n-PB module in the case of the multimodular high-molecular-mass PBPs. Our studies with PBP1 of M. leprae demonstrated that the PB module is capable of binding benzylpenicillin independently of the n-PB module.…”

“…Here we present evidence that a membrane-associating domain, in addition to the non-cleavable pseudo-signal peptide, exists between residues 147 and 314, a feature common to two other high-molecular-mass PBPs of class A, namely PBP1b of E. coli [15,16] and PBP2a of Streptococcus pneumoniae [17]. Unlike PBP1b of E. coli [11], the C-terminal PB module of PBP1 can function as a soluble, penicillin-binding entity independent of the N-terminal non-PB module.…”

“…The high-molecular-mass PBPs of class A are bifunctional enzymes that catalyse both the polymerization of the glycan chains (transglycosylation) and the cross-linking of the peptide chains (transpeptidation) during cell wall synthesis [5][6][7][8][9][10]. The transglycosylation function rests within the N-terminal n-PB module, while the transpeptidation function rests within the C-terminal PB module [11]. In general, the high-molecular-mass PBPs contain near their N-terminus a single hydrophobic transmembrane anchor, which functions as a non-cleavable signal sequence that directs the rest of the protein into the periplasm [12].…”