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...

The potent antibacterial lanthipeptide microvionin, isolated from a culture of Microbacterium arborescens, exhibits a new triamino-dicarboxylic acid moiety, termed avionin, and an unprecedented N-terminal guanidino fatty acid. We identified the corresponding biosynthetic gene cluster and reconstituted central steps of avionin biosynthesis in vitro. Genome mining and isolation of nocavionin from Nocardia terpenica revealed a widespread distribution of this lanthipeptide class, termed lipolanthines, which may be useful as future antimicrobial drugs.

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