Penicillin and Beyond: Evolution, Protein Fold, Multimodular Polypeptides, and Multiprotein Complexes

Ghuysen, Jean‐Marie; Charlier, Paulette; Coyette, Jacques; Duez, Colette; Fonzé, E.; Fraipont, Claudine; Goffin, Colette; Joris, Bernard; Nguyen-Distèche, Martine

doi:10.1089/mdr.1996.2.163

Cited by 40 publications

(21 citation statements)

References 33 publications

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“…That the n-PB module of the class B PBP3 is not a transglycosylase is consistent with the fact that it has a different amino acid signature from that of the n-PB module of the bienzymatic (transglycosylase-transpeptidase) PBP1a and PBP1b of class A and of the monofunctional transglycosylases (12,13,28). In addition to its assisted folding function (16), the n-PB module of PBP3 may serve as a "wiring" element connecting the associated acyl serine transferase module to the transglycosylase module of the class A PBP1a and/or PBP1b and to other cell cycle proteins.…”

Section: Discussionsupporting

confidence: 59%

The bimodular G57-V577 polypeptide chain of the class B penicillin-binding protein 3 of Escherichia coli catalyzes peptide bond formation from thiolesters and does not catalyze glycan chain polymerization from the lipid II intermediate

et al. 1997

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Because the specificity profile of the membrane anchor-free G57-V577 penicillin-binding protein 3 (PBP3) of Escherichia coli for a large series of ␤-lactam antibiotics is similar to that of the full-size membrane-bound PBP, the truncated PBP is expected to adopt the native folded conformation. The truncated PBP3 functions as a thiolesterase. In aqueous media and in the presence of millimolar concentrations of a properly structured amino compound, it catalyzes the aminolysis of the thiolester until completion, suggesting that the penicillinbinding module of PBP3 is designed to catalyze transpeptidation reactions. In contrast, the truncated PBP3 is devoid of glycan polymerization activity on the E. coli lipid II intermediate, suggesting that the non-penicillinbinding module of PBP3 is not a transglycosylase.The multimodular class B penicillin-binding protein 3 (PBP3) is a key element of the cell septation network in Escherichia coli (30). This tripartite protein consists of an M1-E56 membrane anchor-containing module that is fused to a G57-I237 non-penicillin-binding (n-PB) module that is fused to the D238-V577 PB module (7,12,16). The membrane anchor-free G57-V577 polypeptide chain of PBP3 which comprises the n-PB and PB modules has been overproduced in the periplasm of E. coli as an autonomous folding entity (9, 16). With "p" denoting "periplasmic," this truncated PBP3 is called PBP3p.Indirect experimental evidence suggests that the PB module of PBP3 is involved, one way or another, in peptide crosslinking during synthesis of the septal peptidoglycan (3,26). In contrast, the role of the n-PB module of PBP3 is a matter of controversy. According to Ishino and Matsuhashi (19), this module is a transglycosylase catalyzing glycan chain elongation from disaccharide (peptide) lipid II intermediates. According to van Heijenoort et al. (29), it is not.Because the thermostability and affinity for benzylpenicillin and cephalexin of the folded, membrane anchor-free polypeptide are unchanged in comparison with those of the membrane-bound PBP3, one can reasonably postulate that the conformation adopted by the truncated PBP3 reflects faithfully that of the native-state structure (9). ␤-Lactams and thiolester carbonyl donors and the disaccharide (peptide) lipid II intermediate were used to probe the enzymatic activities of the G57-V577 PBP3 and thus shed light on the possible functions of the protein in cell septation. The results are presented below.(Some of the work described in this paper is part of a dissertation presented by M.A. in partial fulfilment of a Ph.D. degree at the University of Liège.) MATERIALS AND METHODSPBP3p and carbonyl donor substrates. PBP3p was purified as described previously (9). It was stored frozen at Ϫ20°C in 10 mM Tris-HCl (pH 8.0)-10% (vol/vol) glycerol-10% (vol/vol) ethylene glycol-0.5 M NaCl. The esters and thiolesters were described previously (1, 2). [14 C]benzylpenicillin (54 Ci/mol) was from Amersham International (Buckinghamshire, United Kingdom). 5Ј-Fluoresceyl-glycyl-6-aminopenicillanate...

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Section: Discussionsupporting

confidence: 59%

The bimodular G57-V577 polypeptide chain of the class B penicillin-binding protein 3 of Escherichia coli catalyzes peptide bond formation from thiolesters and does not catalyze glycan chain polymerization from the lipid II intermediate

et al. 1997

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“…The predicted AmpH protein contains the canonical SXXK, YXN, and KTG activesite motifs of the PBPs and ␤-lactamases (Fig. 4) (15,16). A similarity search using the BEAUTY program (44) indicated that the encoded protein is related most closely to the family of class C ␤-lactamases-15 of the 16 most similar proteins belonged to this group (data not shown).…”

Section: Identification and Subcloning Of Newmentioning

confidence: 98%

AmpC and AmpH, proteins related to the class C beta-lactamases, bind penicillin and contribute to the normal morphology of Escherichia coli

et al. 1997

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Two proteins that bind penicillin were observed in Escherichia coli infected with phages 141, 142, 650, and 651 from the Kohara genomic library. These phages carry chromosomal DNA fragments that do not contain any known penicillin binding protein (PBP) genes, indicating that unrecognized gene products were exhibiting penicillin binding activity. The genes encoding these proteins were subcloned, sequenced, and identified. One gene was ampC, which encodes a chromosomal class C ␤-lactamase. The second gene was located at about 8.5 min on the E. coli genomic map and is a previously uncharacterized open reading frame, here named ampH, that encodes a protein closely related to the class C ␤-lactamases. The predicted AmpH protein is similar in length to AmpC, but there are extensive alterations in the amino acid sequence between the SXXK and YXN motifs of the two proteins. AmpH bound strongly to penicillin G, cefoxitin, and cephalosporin C; was temperature sensitive; and disappeared from cells after overnight incubation in stationary phase. Although closely related to AmpC and other class C ␤-lactamases, AmpH showed no ␤-lactamase activity toward the substrate nitrocefin. Mutation of the ampC and/or ampH genes in E. coli lacking PBPs 1a and 5 produced morphologically aberrant cells, particularly in cell filaments induced by aztreonam. Thus, these two members of the ␤-lactamase family exhibit characteristics similar to those of the classical PBPs, and their absence affects cell morphology. These traits suggest that AmpC and AmpH may play roles in the normal course of peptidoglycan synthesis, remodeling, or recycling.

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“…Each organism contains a complete set of these enzymes, which are all targeted by ␤-lactams. PBPs interact with the antibiotic by forming a relatively stable, covalent complex via the active-site serine (15)(16)(17).…”

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confidence: 99%

All Detectable High-Molecular-Mass Penicillin-Binding Proteins Are Modified in a High-Level β-Lactam-Resistant Clinical Isolate of Streptococcus mitis

Amoroso

Demares

Mollerach

et al. 2001

Antimicrob Agents Chemother

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All detectable high-molecular-mass penicillin-binding proteins (HMM PBPs) are altered in a clinical isolate of Streptococcus mitis for which the ␤-lactam MICs are increased from those previously reported in our region (cefotaxime MIC, 64 g/ml). These proteins were hardly detected at concentrations that saturate all PBPs in clinical isolates and showed, after densitometric analysis, 50-fold-lower radiotracer binding. Resistance was related to mosaic structure in all HMM PBP-coding genes, where critical region replacement was complemented not only by substitutions already reported for the closely related Streptococcus pneumoniae but also by other specific replacements that are presumably close to the active-site serine. Mosaic structure was also presumed in a pbp1a-sensitive strain used for comparison, confirming that these structures do not unambiguously imply, by themselves, detectable critical changes in the kinetic properties of these proteins.

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Penicillin and Beyond: Evolution, Protein Fold, Multimodular Polypeptides, and Multiprotein Complexes

Cited by 40 publications

References 33 publications

The bimodular G57-V577 polypeptide chain of the class B penicillin-binding protein 3 of Escherichia coli catalyzes peptide bond formation from thiolesters and does not catalyze glycan chain polymerization from the lipid II intermediate

The bimodular G57-V577 polypeptide chain of the class B penicillin-binding protein 3 of Escherichia coli catalyzes peptide bond formation from thiolesters and does not catalyze glycan chain polymerization from the lipid II intermediate

AmpC and AmpH, proteins related to the class C beta-lactamases, bind penicillin and contribute to the normal morphology of Escherichia coli

All Detectable High-Molecular-Mass Penicillin-Binding Proteins Are Modified in a High-Level β-Lactam-Resistant Clinical Isolate of Streptococcus mitis

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