Escherichia coli murein-DD-endopeptidase insensitive to beta-lactam antibiotics

Abstract

A novel endopeptidase degrading the pel)tide cross-links in sacculi has been isolated from Escherichia coli and purified to homogeneity. I'lhe enzyme has a molecular weight of 30,000 and, in contrast to already known enzymes of similar specificity, remains fully active in the presence of/3-lactam antibiotics. In adldition, it is exceptional in being inhibited by single-strandetl tleoxyribonucleic acid aild by some polynucleotides. The possible role of the enzymne in cell div-ision is discussed.Hydrolytic enzymes (1,2,6,11,12,18) participate in the metabolism of nmurein sacculi (21) and uindoubtedly p)lay a role in the maintenance of bacterial cell shape. In Escherichia coli, however, the precise biological role of the many murein hydrolases which have been identified remains largely obscure. Among these enzymes, endopeptidases split peptide bridges cross-linking the glycan chains of murein and ar-e strongly inhibited by penicillins, although they are not assumed to be the lethal targets of /3-lactan antibiotics (5,9). I)uring purification of a l)enicillin-sensitive Dr-endopeptidase found to be identical with D-alanine-carboxypeptidase IB (18), we found a novel species of DD-endopeptidase which was totally insensitive to p)enicillin G; some prol)erties of the enzyme have been described previously (20; U. Schwarz, W. Keck, S. Klencke, and H. Mett, Abstr. Svmnip. Finctions Microb. Membranes, 1977). The enzyme thus belongs to a class of hydrolases which remain active in the presence of penicillin and mav be the cause of pericillin-in(duced cell lvsis.In this paper we report on the purification to homogeneity and the p)roperties of the novel endopeptidase. This enzyme is interesting not only because of its biological function but also because it has already been proven to be a powerful analytical tool; we have used it to elucidate the arrangement of the glycan chains within macromolectular sacculi (20). MATERIALS AND METHODSCell extraction and initial enrichment of endopeptidase. The enizyme was isolated fromii E. colli IPA 3092 (14) (iprn Iys), harvested at mid-exponential growth phase, aindl kept frozein at -70C. Cells (2(10 g, wet weight) were openied by shaking with glass beads in a tell mill in 500 mlt of Tris-miialeate (10 imiM; pH (6.8)-10 nmM MgSOi, containing 4(0 pg of DNase per ml (from bovine panc reas -450 Ktnit.z uInits per nmg, Serva). lThe filteired suspeiision (4) was cetntrifuged (6(1 mtmi at 48,0()) x g), the supernatant was (lialyzed three tiies againlst 5 litei-s Of 1'ris-miialeate htffer (1(1 mlM: pHI 5.2). 10 nmM MgSO, and 0.1 miiM dithioetrthritol, cleared by cntitl iftigatioIn (3(1 mIi, 28,0(1 X g (,aid aplplied On a (-olumn (2.6 h)y 31 (cmt; flow rate, :32 mIl/h) of CM Sepharose (I,-6B (IPharmacia Fine Chemiicals eqLilibrated with dialsis btuffer. T'he colunm was eluted with a (ont aeB giadtient (1380 ml of equilibration buffer, :120 ml of 0.5 M KCI in the sainet buffer).En(lopepti(lase was foundl in a hroatd p)eak letwe1tn ().andl (1.3 M K(CI The pooled fraot-tions with endopeptidase were ...

Three redundant murein endopeptidases catalyse an essential cleavage step in peptidoglycan synthesis of Escherichia coliK12

“…On the other hand, Pbp7 is a DD-endopeptidase that is active only on intact murein (Romeis and Holtje, 1994a) and in multiple copies is known to suppress the phenotypic defects of a spr deletion mutant (Hara et al, 1996). Unlike these two Pbps, MepA is a penicillininsensitive DD-endopeptidase (Keck and Schwarz, 1979). However, the absence of these three endopeptidases individually or in combination does not confer any discernible phenotype (Heidrich et al, 2002).…”

“…Pbp4 and Pbp7 are implicated in maintenance of cell shape and cell-cell separation (Meberg et al, 2004;Priyadarshini et al, 2006) and Pbp7 is also believed to be a component of a hypothetical multienzyme complex that functions in murein synthesis (Romeis and Holtje, 1994b). MepA, a penicillin-insensitive, lysostaphin-like-metallopeptidase, is not implicated in any biological process in the cell though its enzymatic activity is very well-studied in vitro (Keck and Schwarz, 1979;van Heijenoort, 2011). AmpH, a lowmolecular-weight penicillin-binding protein has recently been shown to possess both DD-carboxypeptidase and DD-endopeptidase activities (Gonzalez-Leiza et al, 2011).…”

Penicillin‐binding protein 4 of Escherichia coli: molecular cloning of the dacB gene, controlled overexpression, and alterations in murein composition

“…fviurein anaiysis and enzyme assays DO-endopeptidase was assayed in the presence and absence ot 0.2 mM penicillin G using pHJ-Aspm-labelled bisdisaccharidetetrapeptide as substrate (Keck and Schwarz, 1979). The reaction products were quantified after separation by HPLC (Giauner, 1988).…”

Characterization of the proteins encoded by theBacillus subtilis yoxA-dacC  operon

“…It belongs to class C1 of PBPs, also named type 4-PBP in reference to the E. coli PBP4 (Sauvage et al, 2008). The latter is able to cleave the D-alanyl-g-meso-2,6-diaminopimelyl (D-Ala-g-m-A 2 pm) in the peptidoglycan cross-bridges formed by the DD-transpeptidases (Keck & Schwarz, 1979;Korat et al, 1991). This facet of a DD-carboxypeptidase activity is commonly named a 'DD-endopeptidase' activity.…”