References of "Nguyen-Distèche, Martine"
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See detailThe integral membrane FtsW protein and peptidoglycan synthase PBP3 form a subcomplex in Escherichia coli
Fraipont, Claudine ULg; Alexeeva, Svetlana; Wolf, Benoît et al

in Microbiology (2011), 157

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See detailDifferential Functionalities of Amphiphilic Peptide Segments of the Cell-Septation Penicillin-Binding Protein 3 of Escherichia Coli
Marrec-Fairley, Monique; Piette, André ULg; Gallet, Xavier et al

in Molecular Microbiology (2000), 37(5), 1019-1031

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 ... [more ▼]

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. [less ▲]

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See detailRésistance bactérienne aux beta-lactamines
Charlier, Paulette ULg; Coyette, Jacques ULg; Dehareng, Dominique ULg et al

in Medecine Sciences : M/S (1998), 14(5), 544-555

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See detailThe 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
Adam, Maggy; Fraipont, Claudine ULg; Rhazi, Noureddine ULg et al

in Journal of Bacteriology (1997), 179(19), 6005-6009

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 ... [more ▼]

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. [less ▲]

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See detailPenicillin and Beyond: Evolution, Protein Fold, Multimodular Polypeptides, and Multiprotein Complexes
Ghuysen, Jean-Marie ULg; Charlier, Paulette ULg; Coyette, Jacques et al

in Microbial Drug Resistance : Mechanism, Epidemiology, & Disease (1996), 2(2, Summer), 163-175

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 ... [more ▼]

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. [less ▲]

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See detailSite-directed mutagenesis of penicillin-binding protein 3 of Escherichia coli: role of Val-545
Ayala, Juan; Goffin, Colette; Nguyen-Distèche, Martine ULg et al

in FEMS Microbiology Letters (1994), 121(2), 251-256

Val545 of the Escherichia coli penicillin-binding protein 3 is essential to the acyl transfer mechanism through which the active-site serine 307 is acylated by benzylpenicillin and cephalexin and to the ... [more ▼]

Val545 of the Escherichia coli penicillin-binding protein 3 is essential to the acyl transfer mechanism through which the active-site serine 307 is acylated by benzylpenicillin and cephalexin and to the mechanism through which the protein allows rapidly growing cells to divide. [less ▲]

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See detailSite-directed mutagenesis of dicarboxylic acid residues of the penicillin-binding module of the Escherichia coli penicillin-binding protein 3
Goffin, Colette ULg; Ayala, J. A.; Nguyen-Distèche, Martine ULg et al

in FEMS Microbiology Letters (1993), 113(3), 247-251

The glutamic acid E396, aspartic acid D409 and glutamic acid E411 residues of the Escherichia coli penicillin-binding protein 3 were each converted into an alanine residue. As deduced from penicillin ... [more ▼]

The glutamic acid E396, aspartic acid D409 and glutamic acid E411 residues of the Escherichia coli penicillin-binding protein 3 were each converted into an alanine residue. As deduced from penicillin-binding and complementation experiments, none of these dicarboxylic acid residues is involved in the mechanism of acylation by penicillin and none of them is essential for the in vivo functioning of the PBP. The mutation E396, however, causes an increased thermolability of the protein. [less ▲]

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See detailSecretion by Overexpression and Purification of the Water-Soluble Streptomyces K15 Dd-Transpeptidase/Penicillin-Binding Protein
Palomeque-Messia, Pilar; Quittre, Valérie; Leyh-Bouille, Mélina et al

in Biochemical Journal (1992), 288(1), 87-91

Though synthesized with a cleavable signal peptide and devoid of membrane anchors, the 262-amino-acid-residue Streptomyces K15 DD-transpeptidase/penicillin-binding protein is membrane-bound ... [more ▼]

Though synthesized with a cleavable signal peptide and devoid of membrane anchors, the 262-amino-acid-residue Streptomyces K15 DD-transpeptidase/penicillin-binding protein is membrane-bound. Overexpression in Streptomyces lividans resulted in the export of an appreciable amount of the synthesized protein (4 mg/litre of culture supernatant). The water-soluble enzyme was purified close to protein homogeneity with a yield of 75%. It requires the presence of 0.5 M-NaCl to remain soluble. It is indistinguishable from the detergent-extract wild-type enzyme with respect to molecular mass, thermostability, transpeptidase activity and penicillin-binding capacity. [less ▲]

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See detailAmino Acid Sequence of the Penicillin-Binding Protein/DD-Peptidase of Streptomyces K15. Predicted Secondary Structures of the Low Mr Penicillin-Binding Proteins of Class A
Palomeque-Messia, Pilar; Englebert, Serge; Leyh-Bouille, Melina et al

in Biochemical Journal (1991), 279(Pt 1), 223-230

The low-Mr penicillin-binding protein (PBP)/DD-transpeptidase of Streptomyces K15 is synthesized in the form of a 291-amino acid-residue precursor possessing a cleavable 29-amino acid-residue signal ... [more ▼]

The low-Mr penicillin-binding protein (PBP)/DD-transpeptidase of Streptomyces K15 is synthesized in the form of a 291-amino acid-residue precursor possessing a cleavable 29-amino acid-residue signal peptide. Sequence-similarity searches and hydrophobic-cluster analysis show that the Streptomyces K15 enzyme, the Escherichia coli PBPs/DD-carboxy-peptidases 5 and 6, the Bacillus subtilis PBP/DD-carboxypeptidase 5 and the spoIIA product (a putative PBP involved in the sporulation of B. subtilis) are structurally related and form a distinct class A of low-Mr PBPs/DD-peptidases. The distribution of the hydrophobic clusters along the amino acid sequences also shows that the Streptomyces K15 PBP, and by extension the other PBPs of class A, have similarity in the polypeptide folding, with the beta-lactamases of class A, with as reference the Streptomyces albus G and Staphylococcus aureus beta-lactamases of known three-dimensional structure. This comparison allows one to predict most of the secondary structures in the PBPs and the amino acid motifs that define the enzyme active sites. [less ▲]

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See detailAcyltransferase activities of the high-molecular-mass essential penicillin-binding proteins
Adam, Maggy; Damblon, Christian ULg; Jamin, Marc et al

in Biochemical Journal (1991), 279(Part 2), 601-604

The high-molecular-mass penicillin-binding proteins (HMM-PBPs), present in the cytoplasmic membranes of all eubacteria, are involved in important physiological events such as cell elongation, septation or ... [more ▼]

The high-molecular-mass penicillin-binding proteins (HMM-PBPs), present in the cytoplasmic membranes of all eubacteria, are involved in important physiological events such as cell elongation, septation or shape determination. Up to now it has, however, been very difficult or impossible to study the catalytic properties of the HMM-PBPs in vitro. With simple substrates, we could demonstrate that several of these proteins could catalyse the hydrolysis of some thioesters or the transfer of their acyl moiety on the amino group of a suitable acceptor nucleophile. Many of the acyl-donor substrates were hippuric acid or benzoyl-D-alanine derivatives, and their spectroscopic properties enabled a direct monitoring of the enzymic reaction. In their presence, the binding of radioactive penicillin to the PBPs was also inhibited. [less ▲]

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See detailThe Streptomyces K15 DD-peptidase/penicillin-binding protein. Active site and sequence of the N-terminal region.
Leyh-Bouille, Mélina; Vanbeeumen, Jozef; Renier-Pirlot, Suzanne et al

in Biochemical Journal (1989), 260(2), 601-604

The N-terminal region of the Streptomyces K15 DD-peptidase/penicillin-binding protein shows high homology with that of other penicillin-interactive proteins or domains. The active-site serine residue of ... [more ▼]

The N-terminal region of the Streptomyces K15 DD-peptidase/penicillin-binding protein shows high homology with that of other penicillin-interactive proteins or domains. The active-site serine residue of the conserved tetrad Ser-Xaa-Xaa-Lys occurs at position 35. There is no indication for the presence of a signal peptide or an N-terminal hydrophobic sequence, suggesting that the Streptomyces K15 enzyme is probably anchored to the membrane by a C-terminal peptide segment. [less ▲]

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See detailOverexpression, solubilization and refolding of a genetically engineered derivative of the penicillin-binding protein 3 of Escherichia coli K12.
Bartholomé-De Belder, J.; Nguyen-Distèche, Martine ULg; Houba-Herin, N. et al

in Molecular Microbiology (1988), 2(4), 519-525

Replacement of the amino-terminal 40-amino-acid region of the 588-amino-acid precursor of the membrane-bound penicillin-binding protein 3 (PBP3) by the decapeptide MKGKEFQAWI was carried out by altering ... [more ▼]

Replacement of the amino-terminal 40-amino-acid region of the 588-amino-acid precursor of the membrane-bound penicillin-binding protein 3 (PBP3) by the decapeptide MKGKEFQAWI was carried out by altering the amino-coding end of the ftsI gene. Insertion of the modified gene into a runaway-replication plasmid under the control of a fused lpp promoter and lac promoter/operator, resulted in the overexpression by Escherichia coli of the modified PBP3 (designated PBP3**) in the cytoplasm. About 80% of the accumulated PBP3** underwent sequestration in the form of insoluble protein granules that were isolated by cell breakage or cell lysis. After selective removal of contaminants by an EDTA-lysozyme/DNase (deoxyribonuclease)/Nonidet extraction, treatment of the granules with guanidinium chloride followed by dialysis against buffer containing 0.5 M NaCl yielded a refolded, water-soluble PBP3**, which, upon chromatography on Superose 12, exhibited the expected 60,000 molecular mass. The refolded PBP3** bound benzylpenicillin in a 1 to 1 molar ratio, was highly sensitive to aztreonam and showed the same degree of thermostability, in terms of penicillin-binding capacity, as the parent, membrane-bound PBP3, suggesting that protein refolding occurred with formation of the correct intramolecular interactions. Two to three mg of refolded PBP3** can be obtained from 1 litre of culture of the overproducing strain. [less ▲]

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See detailThe pH dependence of the active-site serine DD-peptidase of Streptomyces R61
Varetto, Louis; Frère, Jean-Marie ULg; Nguyen-Distèche, Martine ULg et al

in European Journal of Biochemistry (1987), 162(3), 525-531

Titration of the active-site serine DD-peptidase of Streptomyces R61 shows that formation of acyl enzyme during hydrolysis of the substrate Ac2-L-Lys-D-Ala-D-Ala and enzyme inactivation by the beta-lactam ... [more ▼]

Titration of the active-site serine DD-peptidase of Streptomyces R61 shows that formation of acyl enzyme during hydrolysis of the substrate Ac2-L-Lys-D-Ala-D-Ala and enzyme inactivation by the beta-lactam compounds benzylpenicillin, N-acetylampicillin and ampicillin relies on the acidic form of an enzyme's group of pK approximately equal to 9.5. It is proposed that protonation of a lysine epsilon-amino group facilitates initial binding by charge pairing with the free carboxylate of the substrate and the beta-lactam molecules. Lowering the pH from 7 to 5 has no effect on the second-order rate constant of enzyme acylation by benzylpenicillin and N-acetylampicillin but results in a decreased rate constant of acylation by ampicillin and Ac2-L-Lys-D-Ala-D-Ala. Protonation of the side-chain amino group of ampicillin and a decreased efficacy of the initial binding of the peptide to the enzyme seem to be responsible for the observed effects. Whatever the molecule bound to the enzyme, there is no sign for the active involvement of an enzyme's histidine residue of pK 6.5-7.0 in the hydrolysis pathway. [less ▲]

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See detailEffects of thiol reagents on Streptomyces K15 DD-peptidase-catalysed reactions
Leyh-Bouille, Mélina; Nguyen-Distèche, Martine ULg; Bellefroid-Bourguignon, Catherine et al

in Biochemical Journal (1987), 241(3), 893-897

The 26,000-Mr DD-peptidase of Streptomyces K15 binds one equivalent of thiol reagents as 5,5'-dithiobis-(2-nitrobenzoate) or p-chloromercuribenzoate (pCMB). Derivatization of the DD-peptidase by pCMB ... [more ▼]

The 26,000-Mr DD-peptidase of Streptomyces K15 binds one equivalent of thiol reagents as 5,5'-dithiobis-(2-nitrobenzoate) or p-chloromercuribenzoate (pCMB). Derivatization of the DD-peptidase by pCMB decreases the efficacy of the initial binding of the ester carbonyl donor Ac2-L-Lys-D-Ala-D-lactate to the enzyme (K), the rate of enzyme acylation by the donor (K+2) and the rate of enzyme deacylation (k+3). However, the value of the k+2/k+3 ratio, and therefore the percentage of total enzyme which, at saturating concentrations of the donor, is present as acyl-enzyme at the steady state of the reaction, are not modified. The enzyme's binding sites for pCMB and benzylpenicillin are not mutually exclusive. But, when compared with the native enzyme, the pCMB-derivatized enzyme undergoes acylation by benzylpenicillin with a decreased second-order-rate constant (k+2/K) value and gives rise to a penicilloyl adduct of increased stability. Since the acyl-enzyme mechanism is not annihilated by pCMB derivatization, it is proposed that basically, and like all the other DD-peptidases/penicillin-binding proteins so far characterized, the Streptomyces K15 DD-peptidase is an active-site-serine enzyme. [less ▲]

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See detailActive-site-serine D-alanyl-D-alanine-cleaving-peptidase-catalysed acyl-transfer reactions. Procedures for studying the penicillin-binding proteins of bacterial plasma membranes
Ghuysen, Jean-Marie ULg; Frère, Jean-Marie ULg; Leyh-Bouille, Mélina et al

in Biochemical Journal (1986), 235(1), 159-165

Under certain conditions, the values of the parameters that govern the interactions between the active-site-serine D-alanyl-D-alanine-cleaving peptidases and both carbonyl-donor substrates and beta-lactam ... [more ▼]

Under certain conditions, the values of the parameters that govern the interactions between the active-site-serine D-alanyl-D-alanine-cleaving peptidases and both carbonyl-donor substrates and beta-lactam suicide substrates can be determined on the basis of the amounts of (serine ester-linked) acyl-protein formed during the reactions. Expressing the 'affinity' of a beta-lactam compound for a DD-peptidase in terms of second-order rate constant of enzyme acylation and first-order rate constant of acyl-enzyme breakdown rests upon specific features of the interaction (at a given temperature) and permits study of structure-activity relationships, analysis of the mechanism of intrinsic resistance and use of a 'specificity index' to define the capacity of a beta-lactam compound of discriminating between various sensitive enzymes. From knowledge of the first-order rate constant of acyl-enzyme breakdown and the given time of incubation, the beta-lactam compound concentrations that are necessary to achieve given extents of DD-peptidase inactivation can be converted into the second-order rate constant of enzyme acylation. The principles thus developed can be applied to the study of the multiple penicillin-binding proteins that occur in the plasma membranes of bacteria. [less ▲]

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See detailStreptomyces K15 DD-peptidase-catalysed reactions with suicide beta-lactam carbonyl donors
Leyh-Bouille, Mélina; Nguyen-Distèche, Martine ULg; Pirlot, Suzanne et al

in Biochemical Journal (1986), 235(1), 177-182

The values of the kinetic parameters that govern the interactions between the Streptomyces K15 DD-peptidase and beta-lactam compounds were determined by measuring the inactivating effect that these ... [more ▼]

The values of the kinetic parameters that govern the interactions between the Streptomyces K15 DD-peptidase and beta-lactam compounds were determined by measuring the inactivating effect that these compounds exert on the transpeptidase activity of the enzyme and, in the case of [14C]benzylpenicillin and [14C]cefoxitin, by measuring the amounts of acyl-enzyme formed during the reaction. K15 DD-peptidase binds benzylpenicillin or cefoxitin at a molar ratio of 1:1. Benzylpenicilloate is the major product released during breakdown of the acyl-enzyme formed with benzylpenicillin. Benzylpenicillin is not a better acylating agent than the amide Ac2-L-Lys-D-Ala-D-Ala and ester Ac2-L-Lys-D-Ala-D-lactatecarbonyl-donor substrates. beta-Lactam compounds possessing a methoxy group on the alpha-face of the molecule show high inactivating potency. [less ▲]

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See detailStreptomyces K15 DD-peptidase-catalysed reactions with ester and amide carbonyl donors
Nguyen-Distèche, Martine ULg; Leyh-Bouille, Mélina; Pirlot, Suzanne et al

in Biochemical Journal (1986), 235(1), 167-176

In water, the purified 26 000-Mr membrane-bound DD-peptidase of Streptomyces K15 hydrolyses the ester carbonyl donor Ac2-L-Lys-D-Ala-D-lactate (release of D-lactate) and the amide carbonyl donor Ac2-L-Lys ... [more ▼]

In water, the purified 26 000-Mr membrane-bound DD-peptidase of Streptomyces K15 hydrolyses the ester carbonyl donor Ac2-L-Lys-D-Ala-D-lactate (release of D-lactate) and the amide carbonyl donor Ac2-L-Lys-D-Ala-D-Ala (release of D-alanine) with accumulation of acyl- (Ac2-L-Lys-D-alanyl-)enzyme. Whereas hydrolysis of the ester substrate proceeds to completion, hydrolysis of the amide substrate is negligible because of the capacity of the K15 DD-peptidase for utilizing the released D-alanine in a transfer reaction (Ac2-L-Lys-D-Ala-D-Ala + D-Ala----Ac2-L-Lys-D-Ala-D-Ala + D-Ala) that maintains the concentration of the amide substrate at a constant level. In the presence of an amino acceptor X-NH2 (Gly-Gly or Gly-L-Ala) related to the Streptomyces peptidoglycan, both amide and ester carbonyl donors are processed without detectable accumulation of acyl-enzyme. Under proper conditions, the acceptor activity of water and, in the case of the amide substrate, the acceptor activity of the released D-alanine can be totally overcome so that the two substrates are quantitatively converted into transpeptidated product Ac2-L-Lys-D-Ala-NH-X (and hydrolysis is prevented). Experimental evidence suggests that the amino acceptor modifies both the binding of the carbonyl donor to the enzyme and the ensuing rate of enzyme acylation. [less ▲]

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See detailBacterial wall peptidoglycan, DD-peptidases and beta-lactam antibiotics
Ghuysen, Jean-Marie ULg; Frère, Jean-Marie ULg; Leyh-Bouille, Mélina et al

in Scandinavian Journal of Infectious Diseases (1984), 42

Wall peptidoglycan expansion in bacteria rests upon a cytoplasmic D-Ala: D-Ala ligase (ADP) which catalyses synthesis of a D-Ala-D-Ala dipeptide (with accompanying hydrolysis of one molecule of ATP) and a ... [more ▼]

Wall peptidoglycan expansion in bacteria rests upon a cytoplasmic D-Ala: D-Ala ligase (ADP) which catalyses synthesis of a D-Ala-D-Ala dipeptide (with accompanying hydrolysis of one molecule of ATP) and a set of DD-peptidases which utilize this D-Ala-D-Ala dipeptide--once it has been translocated at the outer face of the plasma membrane as the C-terminal portion of a disaccharide peptide unit--as carbonyl donor for transpeptidation and carboxypeptidation reactions (without additional energy expenditure). Four DD-peptidases have been selected which differ from each other with respect to the effects that amino compounds exert on the fate and rate of consumption of a D-Ala-D-Ala terminated amide carbonyl donor analogue. They serve as models to understand the different mechanisms by which the DD-peptidases perform catalysis and show widely varying responses to the action of beta-lactams, from extreme sensitivity to very high resistance. [less ▲]

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See detailPenicillin-binding proteins and carboxypeptidase/transpeptidase activities in Proteus vulgaris P18 and its penicillin-induced stable L-forms
Rousset, André; Nguyen-Distèche, Martine ULg; Minck, Raymond et al

in Journal of Bacteriology (1982), 152(3), 1042-1048

The originally penicillin-induced, wall-less stable L-forms of Proteus vulgaris P18, isolated by Tulasne in 1949 and since then cultured in he absence of penicillin, have kept the ability to synthesize ... [more ▼]

The originally penicillin-induced, wall-less stable L-forms of Proteus vulgaris P18, isolated by Tulasne in 1949 and since then cultured in he absence of penicillin, have kept the ability to synthesize the seven penicillin-binding proteins and the various DD- and LD-peptidase activities found in the parental bacteria and known to be involved in wall peptidoglycan metabolism. The stable L-forms, however, secrete during growth both the highly penicillin-sensitive, DD-carboxy-peptidase-transpeptidase penicillin-binding protein PBP4 (which in normal bacteria is relatively loosely bound to the plasma membrane) and the penicillin-insensitive LD-carboxypeptidase (which in normal bacteria is located in the periplasmic region). [less ▲]

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See detailIsolation of the membrane-bound 26 000-Mr penicillin-binding protein of Streptomyces strain K15 in the form of a penicillin-sensitive D-alanyl-D-alanine-cleaving transpeptidase
Nguyen-Distèche, Martine ULg; Leyh-Bouille, Mélina; Ghuysen, Jean-Marie ULg

in Biochemical Journal (1982), 207(1), 109-115

The membrane-bound, 26 000-Mr penicillin-binding protein of Streptomyces K15 has been isolated in the form of an effective, penicillin-sensitive D-alanyl-D-alanine-cleaving peptidase exhibiting high ... [more ▼]

The membrane-bound, 26 000-Mr penicillin-binding protein of Streptomyces K15 has been isolated in the form of an effective, penicillin-sensitive D-alanyl-D-alanine-cleaving peptidase exhibiting high transpeptidase activity (greater than 95%) and very low carboxy-peptidase activity (less than 5%). The penicillin-binding protein/transpeptidase can be extracted directly from the mycelium with N-cetyl-NNN-trimethylammonium bromide (Cetavlon) and subsequently obtained at 90% purity and with an 8000-fold specific enrichment (when compared with the activity of the isolated membranes) by a two-step procedure involving Sephadex filtration and affinity chromatography on ampicillin-linked CH Sepharose 4B in the presence of detergent. At saturating concentrations of the co-substrates diacetyl-L-Lys-D-Ala-D-Ala and Gly-Gly, the catalytic-centre activity is about 0.3 s-1. [less ▲]

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