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C Fraipont

Publications and source records attributed to C Fraipont.

10 recordsLinked to original sources
PubMed2000

Differential functionalities of amphiphilic peptide segments of the cell-septation penicillin-binding protein 3 of Escherichia coli.

The class B M1-V577 penicillin-binding protein (PBP) 3 of Escherichia coli consists of a M1-L39 membrane anchor (bearing a cytosolic tail) that is linked via a G40-S70 intervening peptide to an R71-I236 non-catalytic module (containing the conserved motifs 1-3) itself linked via motif 4 to a D237-V577 catalytic module (containing the conserved motifs 5-7 of the penicilloyl serine transferases superfamily). It has been proposed that during cell septation the peptidoglycan crosslinking activity of the acyl transferase module of PBP3 is regulated by the associated M1-I236 polypeptide itself in interaction with other components of the divisome. The fold adopted by the R71-V577 polypeptide of PBP3 has been modelled by reference to the corresponding R76-S634 polypeptide of the class B Streptococcus pneumoniae PBP2x. Based on these data and the results of site-directed mutagenesis of motifs 1-3 and of peptide segments of high amphiphilicity (identified from hydrophobic moment plots), the M1-I236 polypeptide of PBP3 appears to be precisely designed to work in the way proposed. The membrane anchor and the G40-S70 sequence (containing the G57-Q66 peptide segment) upstream from the non-catalytic module have the information ensuring that PBP3 undergoes proper insertion within the divisome at the cell septation site. Motif 1 and the I74-L82 overlapping peptide segment, motif 2 and the H160-G172 overlapping peptide segment, and the G188-D197 motif 3 are located at or close to the intermodule junction. They contain the information ensuring that PBP3 folds correctly and the acyl transferase catalytic centre adopts the active configuration. The E206-V217 peptide segment is exposed at the surface of the non-catalytic module. It has the information ensuring that PBP3 fulfils its cell septation activity within the fully complemented divisome.

Amino Acid Motifs↗
PubMed1998

The structure and function of Escherichia coli penicillin-binding protein 3.

Escherichia coli penicillin-binding protein PBP3 is a key element in cell septation. It is presumed to catalyse a transpeptidation reaction during biosynthesis of the septum peptidoglycan but, in vitro, its enzymatic activity has only been demonstrated with thiolester analogues of the natural peptide substrate. It has no detectable transglycosylase activity with lipid II as substrate. This tripartite protein is constructed of an N-terminal membrane anchor-containing module that is essential for cell septation, a non-penicillin-binding (n-PB) module of unknown function and a C-terminal penicillin-binding (PB) module exhibiting all the characteristic motifs of penicilloyl serine transferases. The n-PB module, which is required for the folding and stability of the PB module, may provide recognition sites for other cell division proteins. Initiation of septum formation is not PBP3-dependent but rests on the appearance of the FtsZ ring, and is thus penicillin-insensitive. The control of PBP3 activity during the cell cycle is briefly discussed.

Amino Acid Sequence↗
PubMed1997

Localization of the Escherichia coli cell division protein Ftsl (PBP3) to the division site and cell pole.

FtsI, also known as penicillin-binding protein 3, is a transpeptidase required for the synthesis of peptidoglycan in the division septum of the bacterium, Escherichia coli. FtsI has been estimated to be present at about 100 molecules per cell, well below the detection limit of immunoelectron microscopy. Here, we confirm the low abundance of FtsI and use immunofluorescence microscopy, a highly sensitive technique, to show that FtsI is localized to the division site during the later stages of cell growth. FtsI was also sometimes observed at the cell pole; polar localization was not anticipated and its significance is not known. We conclude (i) that immunofluorescence microscopy can be used to localize proteins whose abundance is as low as approximately 100 molecules per cell; and (ii) that spatial and temporal regulation of FtsI activity in septum formation is achieved, at least in part, by timed localization of the protein to the division site.

Alkaline Phosphatase↗
PubMed1997

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.

Because the specificity profile of the membrane anchor-free G57-V577 penicillin-binding protein 3 (PBP3) of Escherichia coli for a large series of beta-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 penicillin-binding 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-penicillin-binding module of PBP3 is not a transglycosylase.

Acylation↗
PubMed1996

Penicillin and beyond: evolution, protein fold, multimodular polypeptides, and multiprotein complexes.

As the protein sequence and structure databases expand, the relationships between proteins, the notion of protein superfamily, and the driving forces of evolution are better understood. Key steps of the synthesis of the bacterial cell wall peptidoglycan are revisited in light of these advances. The reactions through which the D-alanyl-D-alanine depeptide is formed, utilized, and hydrolyzed and the sites of action of the glycopeptide and beta-lactam antibiotics illustrate the concept according to which new enzyme functions evolve as a result of tinkering of existing proteins. This occurs by the acquisition of local structural changes, the fusion into multimodular polypeptides, and the association into multiprotein complexes.

Bacterial Proteins↗
PubMed1996

The non-penicillin-binding module of the tripartite penicillin-binding protein 3 of Escherichia coli is required for folding and/or stability of the penicillin-binding module and the membrane-anchoring module confers cell septation activity on the folded structure.

The ftsI-encoded multimodular class B penicillin-binding protein 3 (PBP3) is a key element of the cell septation machinery of Escherichia coli. Altered ftsI genes were overexpressed, and the gene products were analyzed with respect to the level of production, stability, penicillin affinity, and cell septation activity. In contrast to the serine beta-lactamases and low-molecular-mass PBPs which are autonomous folding entities, the S-259-to-V-577 penicillin-binding module of M-1-to-V-577 PBP3 lacks the amino acid sequence information for correct folding. The missing piece of information is provided by the associated G-57-to-E-258 non-penicillin-binding module which functions as a noncleaved, pseudointramolecular chaperone. Key elements of the folding information reside within the motif 1-containing R-60-to-W-110 polypeptide segment and within G-188-to-D-197 motif 3 of the n-PB module. The intermodule interaction is discussed in the light of the known three-dimensional structure (at 3.5-A [0.35-nm] resolution) of the analogous class B PBP2x of Streptococcus pneumoniae (S. Pares, N. Mouz, Y. Pétillot, R. Hakenbeck, and O. Dideberg, Nature Struct. Biol. 3:284-289, 1996). Correct folding and adoption of a stable penicillin-binding conformation are necessary but not sufficient to confer cell septation activity to PBP3 in exponentially growing cells. The in vivo activity of PBP3 also depends on the M-1-to-E-56 amino-terminal module which encompasses the cytosol, the membrane, and the periplasm and which functions as a noncleaved pseudo-signal peptide.

Amino Acid Sequence↗
PubMed1994

Engineering and overexpression of periplasmic forms of the penicillin-binding protein 3 of Escherichia coli.

Replacement of the 36 and 56 N-terminal amino acid residues of the 588-amino-acid-residue membrane-bound penicillin-binding protein 3 (PBP3) of Escherichia coli by the OmpA signal peptide allows export of F37-V577 PBP3 and G57-V577 PBP3 respectively into the periplasm. The modified ftsI genes were placed under the control of the fused lpp promoter and lac promoter/operator; expression of the truncated PBP3s was optimized by varying the copy number of the recombinant plasmids and the amount of LacI repressor, and export was facilitated by increasing the SecB content of the producing strain. The periplasmic PBP3s (yield 8 mg/l of culture) were purified to 70% protein homogeneity. They require the presence of 0.25 M NaCl to remain soluble. Like the membrane-bound PBP3, they undergo processing by elimination of the C-terminal decapeptide I578-S588, they bind penicillin in a 1:1 molar ratio and they catalyse hydrolysis and aminolysis of acyclic thioesters that are analogues of penicillin. The membrane-anchor-free PBP3s have ragged N-termini. The G57-V577 PBP3, however, is less prone to proteolytic degradation than the F37-V577 PBP3.

Amino Acid Sequence↗
PubMed1992

Induction of a Streptomyces cacaoi beta-lactamase gene cloned in S. lividans.

The previously cloned class A beta-lactamase gene (bla) of Streptomyces cacaoi was shown to be inducible by beta-lactam compounds in the host organism S. lividans. A regulatory region of 2.75 kb was identified and the nucleotide sequence determined. It contained four open reading frames (ORFs) of which only two were complete and required for induction. ORF1-ORF2 exerted a positive regulatory effect on the expression of bla. Inactivation of ORF1 or of ORF2 resulted not only in the loss of induction, but also in a 30- to 60-fold decrease in the basal (non-induced) level of beta-lactamase production. ORF1 codes for a DNA-binding protein related to the AmpR repressor/activator, which controls the expression of ampC (class C beta-lactamase) genes in several Enterobacteria.

Amino Acid Sequence↗
PubMed1986

On the origin of bacterial resistance to penicillin: comparison of a beta-lactamase and a penicillin target.

Structural data are now available for comparing a penicillin target enzyme, the D-alanyl-D-alanine-peptidase from Streptomyces R61, with a penicillin-hydrolyzing enzyme, the beta-lactamase from Bacillus licheniformis 749/C. Although the two enzymes have distinct catalytic properties and lack relatedness in their overall amino acid sequences except near the active-site serine, the significant similarity found by x-ray crystallography in the spatial arrangement of the elements of secondary structure provides strong support for earlier hypotheses that beta-lactamases arose from penicillin-sensitive D-alanyl-D-alanine-peptidases involved in bacterial wall peptidoglycan metabolism.

Amino Acid Sequence↗