References of "Frère, Jean-Marie"
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See detailMonitoring the zinc affinity of the metallo-beta-lactamase CphA by automated nanoESI-MS
De Vriendt, K.; Van Driessche, G.; Devreese, B. et al

in Journal of the American Society for Mass Spectrometry (2006), 17(2), 180-188

Metallo-beta-lactamases are zinc containing enzymes that are able to hydrolyze and inactivate beta-lactam antibiotics. The subclass B2 enzyme CphA of Aeromonas hydrophila is a unique metallo-p-lactamase ... [more ▼]

Metallo-beta-lactamases are zinc containing enzymes that are able to hydrolyze and inactivate beta-lactam antibiotics. The subclass B2 enzyme CphA of Aeromonas hydrophila is a unique metallo-p-lactamase because it degrades only carbapenems efficiently and is only active when it has one zinc ion bound. A zinc titration experiment was used to study the zinc affinity of the wild-type and of several mutant CphA enzymes. It shows that a second Zn2+ is also bound at high ion concentrations. All samples were analyzed using mass spectrometry in combination with an automated nanoESI source. The metal-free enzyme has a bimodal charge distribution indicative of two conformational states. A completely folded enzyme is detected when the apo-enzyme has bound the first zinc. Intensity ratios of the different enzyme forms were used to deduce the zinc affinities. CphA enzymes mutated in metal ligands show decreased zinc affinity compared to wild-type, especially D120 mutants. [less ▲]

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See detailCrystal structure of the Mycobacterium fortuitum class A beta-lactamase: structural basis for broad substrate specificity.
Sauvage, Eric ULiege; Fonze, Eveline; Quinting, Birgit et al

in Antimicrobial Agents and Chemotherapy (2006), 50(7), 2516-21

beta-Lactamases are the main cause of bacterial resistance to penicillins and cephalosporins. Class A beta-lactamases, the largest group of beta-lactamases, have been found in many bacterial strains ... [more ▼]

beta-Lactamases are the main cause of bacterial resistance to penicillins and cephalosporins. Class A beta-lactamases, the largest group of beta-lactamases, have been found in many bacterial strains, including mycobacteria, for which no beta-lactamase structure has been previously reported. The crystal structure of the class A beta-lactamase from Mycobacterium fortuitum (MFO) has been solved at 2.13-A resolution. The enzyme is a chromosomally encoded broad-spectrum beta-lactamase with low specific activity on cefotaxime. Specific features of the active site of the class A beta-lactamase from M. fortuitum are consistent with its specificity profile. Arg278 and Ser237 favor cephalosporinase activity and could explain its broad substrate activity. The MFO active site presents similarities with the CTX-M type extended-spectrum beta-lactamases but lacks a specific feature of these enzymes, the VNYN motif (residues 103 to 106), which confers on CTX-M-type extended-spectrum beta-lactamases a more efficient cefotaximase activity. [less ▲]

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See detailSpecificity and reversibility of the transpeptidation reaction catalyzed by the Streptomyces R61 D-Ala-D-Ala peptidase
Rhazi, Noureddine ULiege; Delmarcelle, Michaël ULiege; Sauvage, Eric ULiege et al

in Protein Science (2005), 14(11), 2922-2928

The specificity of the Streptomyces R61 penicillin-sensitive D-Ala-D-Ala peptidase has been re-examined with the help of synthetic substrates. The products of the transpeptidation reactions obtained with ... [more ▼]

The specificity of the Streptomyces R61 penicillin-sensitive D-Ala-D-Ala peptidase has been re-examined with the help of synthetic substrates. The products of the transpeptidation reactions obtained with Gly-L-Xaa dipeptides as acceptor substrates are themselves poor substrates of the enzyme. This is in apparent contradiction with the classically accepted specificity rules for D-Ala-D-Ala peptidases. The Gly-L-Xaa dipeptide is regenerated by both the hydrolysis and transpeptidation reactions. The latter reaction is observed when another Gly-L-Xaa peptide or D-Alanine are supplied as acceptors. Utilization of substrates in which the terminal -COO(-) group has been esterified or amidated shows that a free carboxylate is not an absolute prerequisite for activity. The results are discussed in the context of the expected reversibility of the transpeptidation reaction. [less ▲]

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See detailMolecular characterisation of a versatile peroxidase from a Bjerkandera strain
Moreira, Patricia R.; Duez, Colette ULiege; Dehareng, Dominique ULiege et al

in Journal of Biotechnology (2005), 118(4), 339-352

The cloning and sequencing of the rbpa gene coding for a versatile peroxidase from a novel Bjerkandera strain is hereby reported. The 1777 bp isolated fragment contained a 1698 bp peroxidase-encoding gene ... [more ▼]

The cloning and sequencing of the rbpa gene coding for a versatile peroxidase from a novel Bjerkandera strain is hereby reported. The 1777 bp isolated fragment contained a 1698 bp peroxidase-encoding gene, interrupted by 11 introns. The 367 amino acid-deduced sequence includes a 27 amino acid-signal peptide. The molecular model, built via homology modelling with crystal structures of four fungal peroxidases, highlighted the amino acid residues putatively involved in manganese binding and aromatic substrate oxidation. The potential heme pocket residues (R44, F47, H48, E79, N85, H177, F194 and D239) include both distal and proximal histidines (H48 and H177). RBP possesses potential calcium-binding residues (D49, G67, D69, S71, S178, D195, T197, I200 and D202) and eight cysteine residues (C3, C15, C16, C35, C121, C250, C286, C316). In addition, RIBP includes residues involved in substrate oxidation: three acidic residues (E37, E41 and D183)-putatively involved in manganese binding and H83 and W172-potentially involved in oxidation of aromatic substrates. Characterisation of nucleotide and amino acid sequences include RBP in versatile peroxidase group sharing catalytic properties of both UP and MnP. In addition, the RBP enzyme appears to be closely related with the ligninolytic peroxidases from the Trametes versicolor strain. (C) 2005 Published by Elsevier B.V. [less ▲]

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See detailDramatic broadening of the substrate profile of the Aeromonas hydrophila CphA metallo-beta-lactamase by site-directed mutagenesis
Bebrone, Carine ULiege; Anne, C.; De Vriendt, K. et al

in Journal of Biological Chemistry (2005), 280(31), 28195-28202

Among class B beta-lactamases, the subclass B2 CphA enzyme is characterized by a unique specificity profile. CphA efficiently hydrolyzes only carbapenems. In this work, we generated site-directed mutants ... [more ▼]

Among class B beta-lactamases, the subclass B2 CphA enzyme is characterized by a unique specificity profile. CphA efficiently hydrolyzes only carbapenems. In this work, we generated site-directed mutants that possess a strongly broadened activity spectrum when compared with the WT CphA. Strikingly, the N116H/N220G double mutant exhibits a substrate profile close to that observed for the broad spectrum subclass B1 enzymes. The double mutant is significantly activated by the binding of a second zinc ion under conditions where the WT enzyme is non-competitively inhibited by the same ion. [less ▲]

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See detailNovel use of lipopeptide preparations
Deleu, Magali; Brasseur, Robert; Paquot, Michel et al

Patent (2005)

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See detailDNA vaccination for the priming of neutralizing antibodies against non-immunogenic STa enterotoxin from enterotoxigenic Escherichia coli
Ruth, Nadia ULiege; Mainil, Jacques ULiege; Roupie, Virginie et al

in Vaccine (2005), 23(27), 3618-3627

In order to test the use of DNA vaccination for its capacity to induce antibodies against the non-immunogenic heat-stable enterotoxin STa from Escherichia coli, BALB/c mice were immunized with plasmid DNA ... [more ▼]

In order to test the use of DNA vaccination for its capacity to induce antibodies against the non-immunogenic heat-stable enterotoxin STa from Escherichia coli, BALB/c mice were immunized with plasmid DNA encoding hybrid proteins made by the insertion of wild type STa or insertion of the Cys6Ala, Cys17Ala and Cys6Ala-Cys17Ala STa mutants at positions 195 or 216 of the TEM-1 beta-lactamase. No STa specific antibodies could be detected after three plasmid injections, but a subsequent boost with native STa peptide was capable of inducing low levels of neutralizing antibodies, as tested in the suckling mouse assay. Highest STa specific responses were found in mice primed with the double mutated STa inserted in position 195. This plasmid induced highest T-cell responses to the TEM-1 protein, indicating that priming of helper T-cell responses to the carrier protein was essential. Mixed IgG1/IgG2a isotypes also reflected this T helper 1 type priming. Moreover, insertion into loop A of the TEM-1 carrier may be more suitable than insertion into loop B, because of reduced competition between carrier and hapten B cell responses. [less ▲]

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See detailImproving the alkalophilic performances of the Xyl1 xylanase from Streptomyces sp S38: Structural comparison and mutational analysis
De Lemos Esteves, Frédéric ULiege; Gouders, T.; Lamotte-Brasseur, J. et al

in Protein Science : A Publication of the Protein Society (2005), 14(2), 292-302

Endo-beta-1,4-xylanases of the family 11 glycosyl-hydrolases are catalytically active over a wide range of pH. Xyl1 from Streptomyces sp. S38 belongs to this family, and its optimum pH for enzymatic ... [more ▼]

Endo-beta-1,4-xylanases of the family 11 glycosyl-hydrolases are catalytically active over a wide range of pH. Xyl1 from Streptomyces sp. S38 belongs to this family, and its optimum pH for enzymatic activity is 6. Xyn11 from Bacillus agaradhaerens and XylJ from Bacillus sp. 41M-1 share 85% sequence identity and have been described as highly alkalophilic enzymes. In an attempt to better understand the alkalophilic adaptation of xylanases, the three-dimensional structures of Xyn11 and Xyl1 were compared. This comparison highlighted an increased number of salt-bridges and the presence of more charged residues in the catalytic cleft as well as an eight-residue-longer loop in the alkalophilic xylanase Xyn11. Some of these charges were introduced in the structure of Xyl1 by site-directed mutagenesis with substitutions Y16D, S18E, G50R, N92D, A135Q, E139K, and Y186E. Furthermore, the eight additional loop residues of Xyn11 were introduced in the homologous loop of Xyl1. In addition, the coding sequence of the XylJ catalytic domain was synthesized by recursive PCR, expressed in a Streptomyces host, purified, and characterized together with the Xyl1 mutants. The Y186E substitution inactivated Xyl1, but the activity was restored when this mutation was combined with the G50R or S18E substitutions. Interestingly, the E139K mutation raised the optimum pH of Xyl1 from 6 to 7.5 but had no effect when combined with the N92D substitution. Modeling studies identified the possible formation of an interaction between the introduced lysine and the substrate, which could be eliminated by the formation of a putative salt-bridge in the N92D/E139K mutant. [less ▲]

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See detailA metallo-beta-lactamase enzyme in action: Crystal structures of the monozinc carbapenemase CphA and its complex with biapenem
Garau, Gianpiero; Bebrone, Carine ULiege; Anne, Christine et al

in Journal of Molecular Biology (2005), 345(4), 785-795

One strategy developed by bacteria to resist the action of beta-lactam antibiotics is the expression of metallo-beta-lactamases. CphA from Aeromonas hydrophila is a member of a clinically important ... [more ▼]

One strategy developed by bacteria to resist the action of beta-lactam antibiotics is the expression of metallo-beta-lactamases. CphA from Aeromonas hydrophila is a member of a clinically important subclass of metallo-beta-lactamases that have only one zinc ion in their active site and for which no structure is available. The crystal structures of wild-type CphA and its N220G mutant show the structural features of the active site of this enzyme, which is modeled specifically for carbapenem hydrolysis. The structure of CphA after reaction with a carbapenem substrate, biapenem, reveals that the enzyme traps a reaction intermediate in the active site. These three X-ray structures have allowed us to propose how the enzyme recognizes carbapenems and suggest a mechanistic pathway for hydrolysis of the beta-lactam. This will be relevant for the design of metallo-beta-lactamase inhibitors as well as of antibiotics that escape their hydrolytic activity. (C) 2004 Elsevier Ltd. All rights reserved. [less ▲]

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See detailKinetic properties of four plasmid-mediated AmpC beta-lactamases.
Bauvois, Cedric; Ibuka, Akiko Shimizu; Celso, Almeida et al

in Antimicrobial agents and chemotherapy (2005), 49(10), 4240-6

The heterologous production in Escherichia coli, the purification, and the kinetic characterization of four plasmid-encoded class C beta-lactamases (ACT-1, MIR-1, CMY-2, and CMY-1) were performed. Except ... [more ▼]

The heterologous production in Escherichia coli, the purification, and the kinetic characterization of four plasmid-encoded class C beta-lactamases (ACT-1, MIR-1, CMY-2, and CMY-1) were performed. Except for their instability, these enzymes are very similar to the known chromosomally encoded AmpC beta-lactamases. Their kinetic parameters did not show major differences from those obtained for the corresponding chromosomal enzymes. However, the K(m) values of CMY-2 for cefuroxime, cefotaxime, and oxacillin were significantly decreased compared to those of the chromosomal AmpC enzymes. Finally, the susceptibility patterns of different E. coli hosts producing a plasmid- or a chromosome-encoded class C enzyme toward beta-lactam antibiotics are mainly due to the overproduction of the beta-lactamase in the periplasmic space of the bacteria rather than to a specific catalytic profile of the plasmid-encoded beta-lactamases. [less ▲]

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See detailInactivation of bacterial DD-peptidase by beta-sultams.
Llinas, Antonio; Ahmed, Naveed; Cordaro, Massimiliano et al

in Biochemistry (2005), 44(21), 7738-46

N-Acyl-beta-sultams are time-dependent, irreversible active site-directed inhibitors of Streptomyces R61 DD-peptidase. The rate of inactivation is first order with respect to beta-sultam concentration ... [more ▼]

N-Acyl-beta-sultams are time-dependent, irreversible active site-directed inhibitors of Streptomyces R61 DD-peptidase. The rate of inactivation is first order with respect to beta-sultam concentration, and the second-order rate constants show a dependence on pH similar to that for the hydrolysis of a substrate. Inactivation is due to the formation of a stable 1:1 enzyme-inhibitor complex as a result of the active site serine being sulfonylated by the beta-sultam as shown by ESI-MS analysis and by X-ray crystallography. A striking feature of the sulfonyl enzyme is that the inhibitor is not bound to the oxyanion hole but interacts extensively with the "roof" of the active site where the Arg 285 is located. [less ▲]

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See detailIs it necessary to change the classification of ß-lactamases?
Frère, Jean-Marie ULiege; Galleni, Moreno ULiege; Bush, Karen et al

in Journal of Antimicrobial Chemotherapy (2005), 55(6), 1051-1053

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See detailSpecificity inversion of Ochrobactrum anthropi D-aminopeptidase to a D,D-carboxypeptidase with new penicillin binding activity by directed mutagenesis
Delmarcelle, Michaël ULiege; Boursoit, Marie-Caroline; Filée, Patrice ULiege et al

in Protein Science : A Publication of the Protein Society (2005), 14

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See detailCrystal structure of BRL 42715, C6-(N1-methyl-1,2,3-triazolylmethylene)penem, in complex with Enterobacter cloacae 908R beta-lactamase: evidence for a stereoselective mechanism from docking studies.
Michaux, Catherine; Charlier, Paulette ULiege; Frère, Jean-Marie ULiege et al

in Journal of the American Chemical Society (2005), 127(10), 3262-3

BRL 42715, C6-(N1-methyl-1,2,3-triazolylmethylene)penem, is an active-site-directed inactivator of bacterial beta-lactamases. The crystal structure of Enterobacter cloacae 908R class C beta-lactamase in ... [more ▼]

BRL 42715, C6-(N1-methyl-1,2,3-triazolylmethylene)penem, is an active-site-directed inactivator of bacterial beta-lactamases. The crystal structure of Enterobacter cloacae 908R class C beta-lactamase in complex with BRL 42715, docking, and energy minimization studies explain stereoselectivity of the binding of C6-(heterocyclic methylene)penems against class C beta-lactamase. [less ▲]

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See detailCrystal structure of the Actinomadura R39 DD-peptidase reveals new domains in penicillin-binding proteins.
Sauvage, Eric ULiege; Herman, Raphaël ULiege; Petrella, Stephanie et al

in Journal of Biological Chemistry (2005), 280(35), 31249-56

Actinomadura sp. R39 produces an exocellular DD-peptidase/penicillin-binding protein (PBP) whose primary structure is similar to that of Escherichia coli PBP4. It is characterized by a high beta-lactam ... [more ▼]

Actinomadura sp. R39 produces an exocellular DD-peptidase/penicillin-binding protein (PBP) whose primary structure is similar to that of Escherichia coli PBP4. It is characterized by a high beta-lactam-binding activity (second order rate constant for the acylation of the active site serine by benzylpenicillin: k2/K = 300 mm(-1) s(-1)). The crystal structure of the DD-peptidase from Actinomadura R39 was solved at a resolution of 1.8 angstroms by single anomalous dispersion at the cobalt resonance wavelength. The structure is composed of three domains: a penicillin-binding domain similar to the penicillin-binding domain of E. coli PBP5 and two domains of unknown function. In most multimodular PBPs, additional domains are generally located at the C or N termini of the penicillin-binding domain. In R39, the other two domains are inserted in the penicillin-binding domain, between the SXXK and SXN motifs, in a manner similar to "Matryoshka dolls." One of these domains is composed of a five-stranded beta-sheet with two helices on one side, and the other domain is a double three-stranded beta-sheet inserted in the previous domain. Additionally, the 2.4-angstroms structure of the acyl-enzyme complex of R39 with nitrocefin reveals the absence of active site conformational change upon binding the beta-lactams. [less ▲]

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See detailOn the way of understanding the adaptation to extreme pH of family 11 xylanases: Structural comparison and mutational analysis
De Lemos Esteves, Frédéric ULiege; Ruelle, Virginie; Gouders, Thierry et al

Poster (2004, December)

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See detailGuanidinium chloride denaturation of the dimeric Bacillus licheniformis Blal repressor highlights an independent domain unfolding pathway
Vreuls, Christelle ULiege; Filée, Patrice ULiege; Van Melckebeke, H. et al

in Biochemical Journal (2004), 384(Pt 1), 179-190

The Bacillus licheniformis 74911 BlaI repressor is a prokaryotic regulator that, in the absence of a P-lactam antibiotic, prevents the transcription of the blaP gene, which encodes the BlaP beta-lactamase ... [more ▼]

The Bacillus licheniformis 74911 BlaI repressor is a prokaryotic regulator that, in the absence of a P-lactam antibiotic, prevents the transcription of the blaP gene, which encodes the BlaP beta-lactamase. The BlaI repressor is composed of two structural domains. The 82-residue NTD (N-terminal domain) is a DNA-binding domain, and the CTD (C-terminal domain) containing the next 46 residues is a dimerization domain. Recent studies have shown the existence of the monomeric, dimeric and tetrameric forms of BlaI in solution. In the present study, we analyse the equilibrium unfolding of BlaI in the presence of GdmCl (guanidinium chloride) using different techniques: intrinsic and ANS (8-anilinonaphthalene-1-sulphonic acid) fluorescence, far- and near-UV CD spectroscopy, cross-linking, analytical ultracentrifugation, size exclusion chromatography and NMR spectroscopy. In addition, the intact NTD and CTD were purified after proteolysis of BlaI by papain, and their unfolding by GdmCl was also studied. GdmCl-induced equilibrium unfolding was shown to be fully reversible for BlaI and for the two isolated fragments. The results demonstrate that the NTD and CTD of BlaI fold/unfold independently in a four-step process, with no significant cooperative interactions between them. During the first step, the unfolding of the Blal CTD occurs, followed in the second step by the formation of an 'ANS-bound' intermediate state. Crosslinking and analytical ultracentrifugation experiments suggest that the dissociation of the dimer into two partially unfolded monomers takes place in the third step. Finally, the unfolding of the Blal NTD occurs at a GdmCI concentration of approx. 4 M. In summary, it is shown that the Blal CTD is structured, more flexible and less stable than the NTD upon GdmCI denaturation. These results contribute to the characterization of the Blal dimerization domain (i.e. CTD) involved in the induction process. [less ▲]

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See detailProbing the specificity of the subclass B3 FEZ-1 metallo-beta-lactamase by site-directed mutagenesis
Mercuri, Paola ULiege; Garcia-Saez, I.; De Vriendt, K. et al

in Journal of Biological Chemistry (2004), 279(32), 33630-33638

The subclass B3 FEZ-1 beta-lactamase produced by Fluoribacter (Legionella) gormanii is a Zn(II)-containing enzyme that hydrolyzes the beta-lactam bond in penicillins, cephalosporins, and carbapenems. FEZ ... [more ▼]

The subclass B3 FEZ-1 beta-lactamase produced by Fluoribacter (Legionella) gormanii is a Zn(II)-containing enzyme that hydrolyzes the beta-lactam bond in penicillins, cephalosporins, and carbapenems. FEZ-1 has been extensively studied using kinetic, computational modeling and x-ray crystallography. In an effort to probe residues potentially involved in substrate binding and zinc binding, five site-directed mutants of FEZ-1 (H121A, Y156A, S221A, N225A, and Y228A) were prepared and characterized using metal analyses and steady state kinetics. The activity of H121A is dependent on zinc ion concentration. The H121A monozinc form is less active than the dizinc form, which exhibits an activity similar to that of the wild type enzyme. Tyr156 is not essential for binding and hydrolysis of the substrate. Substitution of residues Ser221 and Asn225 modifies the substrate profile by selectively decreasing the activity against carbapenems. The Y228A mutant is inhibited by the product formed upon hydrolysis of cephalosporins. A covalent bond between the side chain of Cys200 and the hydrolyzed cephalosporins leads to the formation of an inactive and stable complex. [less ▲]

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See detailUpdate of the standard numbering scheme for class B beta-lactamases
Garau, G.; Garcia-Saez, I.; Bebrone, Carine ULiege et al

in Antimicrobial Agents and Chemotherapy (2004), 48(7), 2347-2349

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See detailTwo-color-sum-frequency generation characterization of a protein monolayer adsorbed on a metallic substrate
Dreesen, Laurent ULiege; Sartenaer, Yannick; Humbert, Christophe et al

Poster (2004, June)

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