References of "Frère, Jean-Marie"
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See detailPENICILLIN-BINDING PROTEIN 2X OF STREPTOCOCCUS-PNEUMONIAE - ENZYMATIC-ACTIVITIES AND INTERACTIONS WITH BETA-LACTAMS
JAMIN, M.; Damblon, Christian ULg; MILLIER, S. et al

in Biochemical Journal (1993), 292(Part 3), 735-741

The high-molecular-mass penicillin-binding protein (PBP) 2x, one of the primary targets of beta-lactam antibiotics in Streptococcus pneumoniae, has been produced as a soluble form and purified in large ... [more ▼]

The high-molecular-mass penicillin-binding protein (PBP) 2x, one of the primary targets of beta-lactam antibiotics in Streptococcus pneumoniae, has been produced as a soluble form and purified in large amounts. It has been shown to catalyse hydrolysis and transfer reactions with different ester and thiolester substrates and its catalytic behaviour was often similar to that of the soluble DD-peptidase from Streptomyces R61. This provided an easy method to monitor the activity of the PBP. For the first time, a reliable kinetic study of the interaction between a lethal target and beta-lactam antibiotics has been performed. Characteristic kinetic parameters were obtained with different beta-lactam compounds. These results not only validated the mechanism established with non-essential extracellular enzymes, but will also constitute the basis for comparative studies of the low-affinity variants from penicillin-resistant strains. [less ▲]

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See detailSite-Directed Mutagenesis of the Streptomyces R61 Dd-Peptidase. Catalytic Function of the Conserved Residues around the Active Site and a Comparison with Class-a and Class-C Beta-Lactamases
Hadonou, Ayaovi Medard; Wilkin, Jean-Marc; Varetto, Louis ULg et al

in European Journal of Biochemistry (1992), 207(1), 97-102

The importance of various residues in the Streptomyces R61 penicillin-sensitive DD-peptidase has been assessed by site-directed mutagenesis. The replacement of the active Ser62 by a Cys residue yielded an ... [more ▼]

The importance of various residues in the Streptomyces R61 penicillin-sensitive DD-peptidase has been assessed by site-directed mutagenesis. The replacement of the active Ser62 by a Cys residue yielded an inactive protein which was also unable to recognize penicillin. The activity of the Lys65 → Arg mutant with the peptide and thiol ester substrates was decreased 100-200-fold and the rate of penicillin inactivation was decreased 20 000-fold or more. The mutant thus behaved as a poor, but penicillin-resistant, DD-peptidase. The other studied mutations, the mutations Phe358 → Leu, Tyr90 → Asn, Thr101 → Asn, Phe164 → Ala, Asp225 → Glu and Asp225 → Asn had little influence on the catalytic and penicillin-binding properties. The Asp225 mutants did not exhibit an increased sensitivity to cefotaxime. The Phe164 → Ala mutant was significantly more unstable than the wild-type enzyme. [less ▲]

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See detailPoint Mutations of Two Arginine Residues in the Streptomyces R61 Dd-Peptidase
Bourguignon-Bellefroid, Catherine; Joris, Bernard ULg; Van Beeumen, Jacques et al

in Biochemical Journal (1992), 283(1), 123-128

Incubation of the exocellular DD-carboxypeptidase/transpeptidase of Streptomyces R61 with phenylglyoxal resulted in a time-dependent decrease in the enzyme activity. This inactivation was demonstrated to ... [more ▼]

Incubation of the exocellular DD-carboxypeptidase/transpeptidase of Streptomyces R61 with phenylglyoxal resulted in a time-dependent decrease in the enzyme activity. This inactivation was demonstrated to be due to modification of the Arg-99 side chain. In consequence, the role of that residue was investigated by site-directed mutagenesis. Mutation of Arg-99 into leucine appeared to be highly detrimental to enzyme stability, reflecting a determining structural role for this residue. The conserved Arg-103 residue was also substituted by using site-directed mutagenesis. The modification to a serine residue yielded a stable enzyme, the catalytic properties of which were similar to those of the wild-type enzyme. Thus Arg-103, although strictly conserved or replaced by a lysine residue in most of the active-site penicillin-recognizing proteins, did not appear to fulfil any essential role in either the enzyme activity or structure. [less ▲]

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See detailPrimary and Predicted Secondary Structures of the Actinomadura R39 Extracellular DD-Peptidase, a Penicillin-Binding Protein (PBP) Related to the Escherichia coli PBP4
Granier, Benoît; Duez, Colette ULg; Englebert, Serge et al

in Biochemical Journal (1992), 282(Pt 3), 781-788

As derived from gene cloning and sequencing, the 489-amino-acid DD-peptidase/penicillin-binding protein (PBP) produced by Actinomadura R39 has a primary structure very similar to that of the Escherichia ... [more ▼]

As derived from gene cloning and sequencing, the 489-amino-acid DD-peptidase/penicillin-binding protein (PBP) produced by Actinomadura R39 has a primary structure very similar to that of the Escherichia coli PBP4 [Mottl, Terpstra & Keck (1991) FEMS Microbiol. Lett. 78, 213-220]. Hydrophobic-cluster analysis of the two proteins shows that, providing that a large 174-amino-acid stretch is excluded from the analysis, the bulk of the two polypeptide chains possesses homologues of the active-site motifs and secondary structures found in the class A β-lactamase of Streptomyces albus G of known three-dimensional structure. The 74-amino-acid insert occurs at equivalent places in the two PBPs, between helices α2 and α3, away from the active site. Such an insert is unique among the penicilloyl serine transferases. It is proposed that the Actinomadura R39 PBP and E. coli PBP4 form a special class, class C, of low-Mr PBPs/DD-peptidases. A vector has been constructed and introduced by electrotransformation in the original Actinomadura R39 strain, allowing high-level expression and secretion of the DD-peptidase/PBP (250 mg . 1-1). The gene encoding the desired protein is processed differently in Actinomadura R39 and Streptomyces lividans. Incorrect processing in Streptomyces lividans leads to a secreted protein which is inert in terms of DD-peptidase activity and penicillin-binding capacity. [less ▲]

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See detailImportance of the Two Tryptophan Residues in the Streptomyces R61 Exocellular Dd-Peptidase
Bourguignon-Bellefroid, Catherine; Wilkin, Jean-Marc; Joris, Bernard ULg et al

in Biochemical Journal (1992), 282(Pt 2), 361-367

Modification of the Streptomyces R61 DD-peptidase by N-bromosuccinimide resulted in a rapid loss of enzyme activity. In consequence, the role of the enzyme's two tryptophan residues was investigated by ... [more ▼]

Modification of the Streptomyces R61 DD-peptidase by N-bromosuccinimide resulted in a rapid loss of enzyme activity. In consequence, the role of the enzyme's two tryptophan residues was investigated by site-directed mutagenesis. Trp271 was replaced by Leu. The modification yielded a stable enzyme whose structural and catalytic properties were similar to those of the wild-type protein. Thus the Trp271 residue, though almost invariant among the beta-lactamases of classes A and C and the low-Mr penicillin-binding proteins, did not appear to be essential for enzyme activity. Mutations of the Trp233 into Leu and Ser strongly decreased the enzymic activity, the affinity for beta-lactams and the protein stability. Surprisingly, the benzylpenicilloyl-(W233L)enzyme deacylated at least 300-fold more quickly than the corresponding acyl-enzyme formed with the wild-type protein and gave rise to benzylpenicilloate instead of phenylacetylglycine. This mutant DD-peptidase thus behaved as a weak beta-lactamase. [less ▲]

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See detailImportance of the His-298 Residue in the Catalytic Mechanism of the Streptomyces R61 Extracellular Dd-Peptidase
Hadonou, Ayaovi M.; Jamin, Marc; Adam, Maggy et al

in Biochemical Journal (1992), 282(Pt 2), 495-500

Among the active-site-serine penicillin-recognizing proteins, the Streptomyces R61 extracellular DD-peptidase is the only one to have a His-Thr-Gly sequence [instead of Lys-Thr(Ser)-Gly] in 'box' VII. The ... [more ▼]

Among the active-site-serine penicillin-recognizing proteins, the Streptomyces R61 extracellular DD-peptidase is the only one to have a His-Thr-Gly sequence [instead of Lys-Thr(Ser)-Gly] in 'box' VII. The His residue was replaced by Gln or Lys. Both mutations induced a marked decrease in the rates of both tripeptide substrate hydrolysis and acylation by benzylpenicillin and cephalosporin C. The rate of hydrolysis of the thioester hippuryl thioglycollate was less affected. The most striking result was the disproportionate loss of transpeptidation properties by both mutants, indicating an important role of His-298 in this reaction. We believe that this result represents the first modification of a DD-peptidase leading to a specific decrease of the transpeptidation-to-hydrolysis ratio. [less ▲]

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See detailStreptomyces albus G serine β-lactamase. Probing of the catalytic mechanism via molecular modelling of mutant enzymes
Lamotte-Brasseur, Josette; Jacob-Dubuisson, Françoise; Dive, Georges ULg et al

in Biochemical Journal (1992), 282(Pt 1), 189-195

In previous studies, several amino acids of the active site of class A , β-lactamases have been modified by site-directed mutagenesis. On the basis of the catalytic mechanism proposed for the Streptomyces ... [more ▼]

In previous studies, several amino acids of the active site of class A , β-lactamases have been modified by site-directed mutagenesis. On the basis of the catalytic mechanism proposed for the Streptomyces albus G , β-lactamase [Lamotte- Brasseur, Dive, Dideberg, Charlier, Frere & Ghuysen (1991) Biochem. J. 279, 213-221], the influence that these mutations exert on the hydrogen-bonding network of the active site has been analysed by molecular mechanics. The results satisfactorily explain the effects of the mutations on the kinetic parameters of the enzyme's activity towards a set of substrates. The present study also shows that, upon binding a properly structured ,β-lactam compound, the impaired cavity of a mutant enzyme can readopt a functional hydrogen-bonding-network configuration. [less ▲]

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See detailSynthèse, étude théorique et évaluation biologique de dérivés du 4-amino-4H-1,2,4-triazole analogues des antibiotiques b-lactamiques
Pirotte, Bernard ULg; Dive, Georges ULg; Delarge, Jacques et al

in European Journal of Medicinal Chemistry (1992), 27(3), 193-205

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See detailAnomalous Behaviour of a Protein During Sds/Page Corrected by Chemical Modification of Carboxylic Groups
Matagne, André ULg; Joris, Bernard ULg; Frère, Jean-Marie ULg

in Biochemical Journal (1991), 280((Pt 2)), 553-6

The 29,000-Mr Actinomadura R39 beta-lactamase exhibited a remarkably low electrophoretic mobility on SDS/PAGE, yielding an Mr value almost twice that computed from the corresponding gene sequence. We ... [more ▼]

The 29,000-Mr Actinomadura R39 beta-lactamase exhibited a remarkably low electrophoretic mobility on SDS/PAGE, yielding an Mr value almost twice that computed from the corresponding gene sequence. We showed that chemical modification of the carboxylic groups of glutamic acid and aspartic acid residues restored a normal electrophoretic mobility and that the anomalous behaviour of that protein on SDS/PAGE was due to its very large negative charge at neutral pH. We also compared the behaviour of the same enzyme on gel filtration in the presence of SDS with those of other class A beta-lactamases (Mr approx. 30,000). These experiments suggested that the very low electrophoretic mobility of the Actinomadura R39 beta-lactamase upon SDS/PAGE was more probably due to a low degree of SDS binding rather than to an unusual shape of the SDS-protein complex. [less ▲]

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See detailComparison of the Sequences of Class a β-lactamase and of the Secondary Structure Elements of Penicillin-Recognizing Proteins
Joris, Bernard ULg; Ledent, P.; Dideberg, O. et al

in Antimicrobial Agents and Chemotherapy (1991), 35(11), 2294-2301

The sequences of class A beta-lactamases were compared. Four main groups of enzymes were distinguished: those from the gram-negative organisms and bacilli and two distinct groups of Streptomyces spp. The ... [more ▼]

The sequences of class A beta-lactamases were compared. Four main groups of enzymes were distinguished: those from the gram-negative organisms and bacilli and two distinct groups of Streptomyces spp. The Staphylococcus aureus PC1 enzyme, although somewhat closer to the enzyme from the Bacillus group, did not belong to any of the groups of beta-lactamases. The similarities between the secondary structure elements of these enzymes and those of the class C beta-lactamases and of the Streptomyces sp. strain R61 DD-peptidase were also analyzed and tentatively extended to the class D beta-lactamases. A unified nomenclature of secondary structure elements is proposed for all the penicillin-recognizing enzymes. [less ▲]

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See detailMechanism of acyl transfer by the class A serine β-lactamase of Streptomyces albus G
Lamotte-Brasseur, Josette; Dive, Georges ULg; Dideberg, Otto et al

in Biochemical Journal (1991), 279(Pt 1), 213-221

Optimization by energy minimization of stable complexes occurring along the pathway of hydrolysis of benzylpenicillin and cephalosporin C by the Streptomyces albus G beta-lactamase has highlighted a ... [more ▼]

Optimization by energy minimization of stable complexes occurring along the pathway of hydrolysis of benzylpenicillin and cephalosporin C by the Streptomyces albus G beta-lactamase has highlighted a proton shuttle that may explain the catalytic mechanism of the beta-lactamases of class A. Five residues, S70, S130, N132, T235 and A237, are involved in ligand binding. The gamma-OH group of T235 and, in the case of benzylpenicillin, the gamma-OH group of S130 interact with the carboxylate group, on one side of the ligand molecule. The side-chain NH2 group of N132 and the carbonyl backbone of A237 interact with the exocyclic CONH amide bond, on the other side of the ligand. The backbone NH groups of S70 and A237 polarize the carbonyl group of the scissile beta-lactam amide bond. Four residues, S70, K73, S130 and E166, and two water molecules, W1 and W2, perform hydrolysis of the bound beta-lactam compound. E166, via W1, abstracts the proton from the gamma-OH group of S70. While losing its proton, the O-gamma atom of S70 attacks the carbonyl carbon atom of the beta-lactam ring and, concomitantly, the proton is delivered back to the adjacent nitrogen atom via W2, K73 and S130, thus achieving formation of the acyl-enzyme. Subsequently, E166 abstracts a proton from W1. While losing its proton, W1 attacks the carbonyl carbon atom of the S70 ester-linked acyl-enzyme and, concomitantly, re-entry of a water molecule W'1 replacing W1 allows E166 to deliver the proton back to the same carbonyl carbon atom, thus achieving hydrolysis of the beta-lactam compound and enzyme recovery. The model well explains the differences found in the kcat. values for hydrolysis of benzylpenicillin and cephalosporin C by the Streptomyces albus G beta-lactamase. It also explains the effects caused by site-directed mutagenesis of the Bacillus cereus beta-lactamase I [Gibson, Christensen [less ▲]

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See detailThe Importance of the Negative Charge of Beta-Lactam Compounds in the Interactions with Active-Site Serine Dd-Peptidases and Beta-Lactamases
Varetto, Louis ULg; De Meester, Fabien; Monnaie, Didier et al

in Biochemical Journal (1991), 278(Pt 3), 801-807

The interaction between various penicillins and cephalosporins the carboxylate group of which at C-3 or C-4 had been esterified or amidated and different penicillin-recognizing enzymes was studied. In ... [more ▼]

The interaction between various penicillins and cephalosporins the carboxylate group of which at C-3 or C-4 had been esterified or amidated and different penicillin-recognizing enzymes was studied. In general, our findings reinforced the common assumption that an anionic group at that position is necessary for the effective acylation of these enzymes. However, the relative activities of the modified beta-lactams as inactivators of the Streptomyces R61 DD-peptidase or as substrates of the Bacillus licheniformis, Streptomyces albus G and Enterobacter cloacae beta-lactamases did not fit a general scheme in which the intrinsic electronic and geometric properties of the beta-lactam compounds would be sufficient to explain their substrate or inactivator properties towards the various types of enzymes investigated. [less ▲]

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See detailActive-Site Serine Mutants of the Streptomyces Albus G Beta-Lactamase
Jacob, Françoise; Joris, Bernard ULg; Frère, Jean-Marie ULg

in Biochemical Journal (1991), 277((Pt 3)), 647-52

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces ... [more ▼]

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type. [less ▲]

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See detailA standard numbering scheme for the class A beta-lactamases
Ambler, R. P.; Coulson, A. F. W.; Frère, Jean-Marie ULg et al

in Biochemical Journal (1991), 276(Pt 1), 269-270

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See detailRagged N-Termini and Other Variants of Class a Beta-Lactamases Analysed by Chromatofocusing
Matagne, André ULg; Joris, Bernard ULg; Van Beeumen, J. et al

in Biochemical Journal (1991), 273(273), 503-10

Four beta-lactamases excreted by Gram-positive bacteria exhibited microheterogeneity when analysed by chromatofocusing or ion-exchange chromatography. Ragged N-termini were in part responsible for the ... [more ▼]

Four beta-lactamases excreted by Gram-positive bacteria exhibited microheterogeneity when analysed by chromatofocusing or ion-exchange chromatography. Ragged N-termini were in part responsible for the charge variants, but deamidation of an asparagine residue was also involved, at least for the Bacillus licheniformis enzyme. The activity of a contaminating proteinase could also be demonstrated in the case of Actinomadura R39 beta-lactamase. With that enzyme, proteolysis resulted in partial inactivation, but the inactivated fragments were easily separated from the active forms. With these, as with the other enzymes, the kinetic parameters of the major variants were identical with those of the mixture within the limits of experimental error, so that the catalytic properties of these enzymes can be determined with the 'heterogeneous' preparations. [less ▲]

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See detailMechanism of action of β-lactamases and DD-peptidases
Frère, Jean-Marie ULg; Joris, Bernard ULg; Jacob, Françoise et al

in Pandit, U.K.; Alderweireldt, F.C. (Eds.) Bioorganic Chemistry in Healthcare and Technology (1991)

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See detailCloning and nucleotide sequencing of the gene encoding the beta-lactamase from Citrobacter diversus
Perilli, Mariagrazia; Franceschini, Nicola; Segatore, Bernardetta et al

in FEMS Microbiology Letters (1991), 83

The gene coding for the class A beta-lactamase of Citrobacter diversus has been cloned and sequenced. It contains the information for a 294-amino-acid precursor protein, including a 27-residue N-terminal ... [more ▼]

The gene coding for the class A beta-lactamase of Citrobacter diversus has been cloned and sequenced. It contains the information for a 294-amino-acid precursor protein, including a 27-residue N-terminal signal peptide. The deduced sequence of the N-terminal portion of the mature protein is in excellent agreement with that determined by microsequencing of the protein and readily explains the pI differences observed between the naturally occurring forms I and II of the enzyme. The sequence of the mature protein exhibits a very high degree of similarity with that of the Klebsiella oxytoca class A beta-lactamase [less ▲]

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See detailTHE MUTATION LYS234HIS YIELDS A CLASS-A BETA-LACTAMASE WITH A NOVEL PH-DEPENDENCE
BRANNIGAN, J.; Matagne, André ULg; Jacob, Françoise et al

in Biochemical Journal (1991), 278(Part 3), 673-678

The lysine-234 residue is highly conserved in beta-lactamases and in nearly all active-site-serine penicillin-recognizing enzymes. Its replacement by a histidine residue in the Streptomyces albus G class ... [more ▼]

The lysine-234 residue is highly conserved in beta-lactamases and in nearly all active-site-serine penicillin-recognizing enzymes. Its replacement by a histidine residue in the Streptomyces albus G class A beta-lactamase yielded an enzyme the pH-dependence of which was characterized by the appearance of a novel pK, which could be attributed to the newly introduced residue. At low pH, the k(cat.) value for benzylpenicillin was as high as 50 % of that of the wild-type enzyme, demonstrating that an efficient active site was maintained. Both k(cat.) and k(cat.)/K(m) dramatically decreased above pH 6 but the decrease in k(cat.)/K(m) could not be attributed to larger K(m) values. Thus a positive charge on the side chain of residue 234 appears to be more essential for transition-state stabilization than for initial recognition of the substrate ground state. [less ▲]

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See detailDiversity of the Mechanisms of Resistance to Beta-Lactam Antibiotics
Frère, Jean-Marie ULg; Joris, Bernard ULg; Granier, B. et al

in Research in Microbiology (1991), 142(6, Jul-Aug), 705-10

The sensitivity of a bacterium to beta-lactam antibiotics depends upon the interplay between 3 independent factors: the sensitivity of the essential penicillin-binding enzyme(s), the quantity and ... [more ▼]

The sensitivity of a bacterium to beta-lactam antibiotics depends upon the interplay between 3 independent factors: the sensitivity of the essential penicillin-binding enzyme(s), the quantity and properties of the beta-lactamase(s) and the diffusion barrier that the outer-membrane of Gram-negative bacteria can represent. Those three factors can be modified by mutations or by the horizontal transfer of genes or portions of genes. [less ▲]

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See detailAntibacterial activity of 5-acylaminothiazole derivatives, synthetic drugs related to β-lactam antibiotics
Pirotte, Bernard ULg; Delarge, Jacques; Coyette, Jacques ULg et al

in Journal of Antibiotics (The) (1991), 44

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