References of "Frère, Jean-Marie"
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See detailThe Precursor of the Streptomyces R61 Dd-Peptidase Containing a C-Terminal Extension Is Inactive
Fanuel, Laurence; Granier, Benoît; Wilkin, Jean-Marc et al

in FEBS Letters (1994), 351(1), 49-52

The Streptomyces R61 DD-peptidase gene encodes a 26-residue C-terminal extension which is not found in the mature protein. When the gene was expressed in Escherichia coli, the extension was not cleaved ... [more ▼]

The Streptomyces R61 DD-peptidase gene encodes a 26-residue C-terminal extension which is not found in the mature protein. When the gene was expressed in Escherichia coli, the extension was not cleaved and the precursor protein was not enzymatically active. It also reacted with penicillins significantly more slowly than the mature protein. The introduction of a 'stop' codon after that corresponding to the C-terminal residue of the mature protein resulted in the production of an active protein in the periplasm of E. coli. [less ▲]

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See detailThe mechanism of action of DD-peptidases: the role of Threonine-299 and -301 in the Streptomyces R61 DD-peptidase.
Wilkin, J M; Dubus, Alice ULg; Joris, Bernard ULg et al

in Biochemical Journal (1994), 301 ( Pt 2)

The side chains of residues Thr299 and Thr301 in the Streptomyces R61 DD-peptidase have been modified by site-directed mutagenesis. These amino acids are part of a beta-strand which forms a wall of the ... [more ▼]

The side chains of residues Thr299 and Thr301 in the Streptomyces R61 DD-peptidase have been modified by site-directed mutagenesis. These amino acids are part of a beta-strand which forms a wall of the active-site cavity. Thr299 corresponds to the second residue of the Lys-Thr(Ser)-Gly triad, highly conserved in active-site beta-lactamases and penicillin-binding proteins (PBPs). Modification of Thr301 resulted only in minor alterations of the catalytic and penicillin-binding properties of the enzyme. No selective decrease of the rate of acylation was observed for any particular class of compounds. By contrast, the loss of the hydroxy group of the residue in position 299 yielded a seriously impaired enzyme. The rates of inactivation by penicillins were decreased 30-50-fold, whereas the reactions with cephalosporins were even more affected. The efficiency of hydrolysis against the peptide substrate was also seriously decreased. More surprisingly, the mutant was completely unable to catalyse transpeptidation reactions. The conservation of an hydroxylated residue in this position in PBPs is thus easily explained by these results. [less ▲]

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See detailTranscription and expression analysis, using lacZ and phoA gene fusions, of Mycobacterium fortuitum beta-lactamase genes cloned from a natural isolate and a high-level beta-lactamase producer.
Timm, J; Perilli, M G; Duez, Colette ULg et al

in Molecular Microbiology (1994), 12(3), 491-504

The gene encoding a class A beta-lactamase was cloned from a natural isolate of Mycobacterium fortuitum (blaF) and from a high-level amoxicillin-resistant mutant that produces large amounts of beta ... [more ▼]

The gene encoding a class A beta-lactamase was cloned from a natural isolate of Mycobacterium fortuitum (blaF) and from a high-level amoxicillin-resistant mutant that produces large amounts of beta-lactamase (blaF*). The nucleotide sequences of the two genes differ at 11 positions, including two in the region upstream from the coding sequence. Gene fusions to Escherichia coli lacZ and transcription and expression analysis of the cloned genes in Mycobacterium smegmatis indicated that high-level production of the beta-lactamase in the mutant is mainly or wholly due to a single base pair difference in the promoter. These analyses also showed that transcription and translation start at the same position. A comparison of the amino acid sequence of BlaF, as predicted from the nucleotide sequence, with the determined N-terminal amino acid sequence indicated the presence of a typical signal peptide. The fusion of blaF (or blaF*) to the E. coli gene phoA resulted in the production of BlaF-PhoA hybrid proteins that had alkaline phosphatase activity. These results demonstrate that phoA can be used as a reporter gene for studying protein export in mycobacteria. [less ▲]

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See detailAnalysis of the penA gene of Pseudomonas cepacia 249.
Joris, Bernard ULg; Galleni, Moreno ULg; Frère, Jean-Marie ULg et al

in Antimicrobial Agents and Chemotherapy (1994), 38(2), 407-8

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See detailA common system controls the induction of very different genes. The class-A beta-lactamase of Proteus vulgaris and the enterobacterial class-C beta-lactamase.
Datz, M; Joris, Bernard ULg; Azab, E A et al

in European Journal of Biochemistry (1994), 226(1), 149-57

Among the Enterobacteriaceae, Proteus vulgaris is exceptional in the inducible production of a 29-kDa beta-lactamase (cefuroximase) with an unusually high activity towards the beta-lactamase-stable ... [more ▼]

Among the Enterobacteriaceae, Proteus vulgaris is exceptional in the inducible production of a 29-kDa beta-lactamase (cefuroximase) with an unusually high activity towards the beta-lactamase-stable oximino-cephalosporins (e.g. cefuroxime and cefotaxime). Sequencing of the corresponding gene, cumA, showed that the derived CumA beta-lactamase belonged to the molecular class A. The structural gene was under the direct control of gene cumR, which was transcribed backwards and whose initiation codon was 165 bp away from that of the beta-lactamase gene. This resembled the arrangement of structural and regulator genes ampC and ampR of the 39-kDa molecular-class-C beta-lactamase AmpC present in many enterobacteria. Moreover, cloned genes ampD and ampG for negative modulation and signal transduction of AmpC beta-lactamase induction, respectively, were also able to restore constitutively CumA overproducing and non-inducible P. vulgaris mutants to the inducible, wild-type phenotype. The results indicate that controls of the induction phenomena are equivalent for the CumA and AmpC beta-lactamase. Very different structural genes can thus be under the control of identical systems. [less ▲]

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See detailSerine-Type D-Ala-D-Ala Peptidases and Penicillin-Binding Proteins
Granier, Benoît; Jamin, Marc; Adam, Maggy et al

in Methods in Enzymology (1994), 244

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See detailSTRUCTURAL AND KINETIC CHARACTERIZATION OF A BETA-LACTAMASE-INHIBITOR PROTEIN
STRYNADKA, N. C. J.; JENSEN, S. E.; JOHNS, K. et al

in Nature (1994), 368(6472), 657-660

THE past decade has seen an alarming worldwide increase in resistance to beta-lactam antibiotics among many pathogenic bacteria1, which is due mainly to plasmid- or chromosomally encoded beta-lactamases ... [more ▼]

THE past decade has seen an alarming worldwide increase in resistance to beta-lactam antibiotics among many pathogenic bacteria1, which is due mainly to plasmid- or chromosomally encoded beta-lactamases that specifically cleave penicillin and cephalosporins, rendering them inactive. There is therefore a need to develop new strategies in the design of effective inhibitors of beta-lactamase. All the small-molecule inhibitors in clinical use are not very effective and are rapidly degraded2,3. Furthermore, newly characterized mutants of the plasmid-mediated beta-lactamase TEM-1 are highly resistant to these small-molecule inhibitors, including clavulanic acid and tazobactam4. It has been shown that Streptomyces clavuligerus produces an exocellular beta-lactamase inhibitory protein (BLIP; M(r) 17.5 K)5. Here we present data defining BLIP as the most effective known inhibitor of a variety of beta-lactamases, with K(i) values in the subnanomolar to picomolar range. To identify those features in BLIP that make it such a potent inhibitor, we have determined its molecular structure at 2.1 angstrom resolution. BLIP is a relatively flat molecule with a unique fold, comprising a tandem repeat of a 76-amino-acid domain. Each domain consists of a helix-loop-helix motif that packs against a four-stranded antiparallel beta-sheet (Fig. 1a). To our knowledge, BLIP is the first example of a protein inhibitor having two similarly folded domains that interact with and inhibit a single target enzyme. [less ▲]

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See detailDirect n.m.r. evidence for substrate-induced conformational changes in a beta-lactamase.
Jamin, M.; Damblon, Christian ULg; Bauduin-Misselyn, A. M. et al

in Biochemical Journal (1994), 301 ( Pt 1)

Cefoxitin and other beta-lactam antibiotics with a methoxy group on the alpha-face behave as very poor substrates of the Bacillus licheniformis beta-lactamase. The kinetic properties of the enzyme ... [more ▼]

Cefoxitin and other beta-lactam antibiotics with a methoxy group on the alpha-face behave as very poor substrates of the Bacillus licheniformis beta-lactamase. The kinetic properties of the enzyme-cefoxitin system made it theoretically suitable for a detailed structural study of the acyl-enzyme. Unfortunately, soaking the crystals in cefoxitin solution did not allow detection of a crystalline acyl-enzyme complex. In contrast, direct observation by n.m.r. of the stable acyl-enzyme formed with cefoxitin and moxalactam indicated clear modifications of the enzyme structure, which were reflected in the aromatic and high-field methyl regions of the spectrum. The return to the initial free enzyme spectrum was concomitant with the hydrolysis of the acyl-enzyme, the process being slow enough to allow multidimensional n.m.r. experiments. [less ▲]

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See detailThe structures and catalytic mechanisms of active-site serine beta-lactamases.
Lamotte, Josette ULg; Knox, J.; Kelly, J. A. et al

in Biotechnology & Genetic Engineering Reviews (1994), 12

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See detailInteractions between Active-Site-Serine Beta-Lactamases and Mechanism-Based Inactivators: A Kinetic Study and an Overview
Matagne, André ULg; Ghuysen, M. F.; Frère, Jean-Marie ULg

in Biochemical Journal (1993), 295((Pt 3)), 705-711

The interactions between three class A beta-lactamases and three beta-lactamase inactivators (clavulanic acid, sulbactam and olivanic acid MM13902) were studied. Interestingly, the interaction between the ... [more ▼]

The interactions between three class A beta-lactamases and three beta-lactamase inactivators (clavulanic acid, sulbactam and olivanic acid MM13902) were studied. Interestingly, the interaction between the Streptomyces cacaoi beta-lactamase and clavulanate indicated little irreversible inactivation. With sulbactam, irreversible inactivation was found to occur with the three studied enzymes, but no evidence for transiently inactivated adducts was found. Irreversible inactivation of the S. albus G and S. cacaoi enzymes was particularly slow. With olivanate, irreversible inactivation was also observed with the three enzymes, but with the S. cacaoi enzyme, no hydrolysis could be detected. A tentative summary of the results found in the literature is also presented (including 6 beta-halogenopenicillanates), and the general conclusions underline the diversity of the mechanisms and the wide variations of the rate constants observed when class A beta-lactamases interact with beta-lactamase inactivators, in agreement with the behaviours of the same enzymes towards their good and poor substrates. [less ▲]

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See detailInteractions between Active-Site Serine Beta-Lactamases and So-Called Beta-Lactamase-Stable Antibiotics. Kinetic and Molecular Modelling Studies
Matagne, André ULg; Lamotte-Brasseur, J.; Frère, Jean-Marie ULg

in European Journal of Biochemistry (1993), 217(1), 61-67

The interactions between imipenem and four monobactams and three class A beta-lactamases have been studied in detail. Despite their reputation as being beta-lactamase-stable, some of these compounds were ... [more ▼]

The interactions between imipenem and four monobactams and three class A beta-lactamases have been studied in detail. Despite their reputation as being beta-lactamase-stable, some of these compounds were significantly hydrolysed by the enzymes. The results obtained with the Streptomyces albus G beta-lactamase have been analysed in the light of molecular modelling studies. The discussion is extended to include other so-called beta-lactamase-stable antibiotics to demonstrate that this appellation can often be misleading. [less ▲]

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See detailInteractions between Active-Site-Serine Beta-Lactamases and Compounds Bearing a Methoxy Side Chain on the Alpha-Face of the Beta-Lactam Ring: Kinetic and Molecular Modelling Studies
Matagne, André ULg; Lamotte-Brasseur, J.; Dive, Georges ULg et al

in Biochemical Journal (1993), 293((Pt 3)), 607-611

The interactions between three class A beta-lactamases and compounds bearing a methoxy side chain on the alpha-face of the beta-lactam ring (cefoxitin, moxalactam and temocillin) have been studied. When ... [more ▼]

The interactions between three class A beta-lactamases and compounds bearing a methoxy side chain on the alpha-face of the beta-lactam ring (cefoxitin, moxalactam and temocillin) have been studied. When compared with the situation prevailing with good substrates, both acylation and deacylation steps appeared to be severely impaired. Molecular modelling studies of the structures of the Henri-Michaelis complexes and of the acyl-enzymes indicate a major displacement of the crystallographically observed water molecule which connects the glutamate-166 and serine-70 side chains and underline the role of this water molecule in both reaction steps. [less ▲]

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See detailThe Mechanism of Action of DD-Peptidases: The Role of Tyrosine-159 in the Streptomyces R61 DD-Peptidase
Wilkin, Jean-Marc; Jamin, Marc; Damblon, Christian ULg et al

in Biochemical Journal (1993), 291(Part 2), 537-544

Tyrosine-159 of the Streptomyces R61 penicillin-sensitive DD-peptidase was replaced by serine or phenylalanine. The second mutation yielded a very poorly active protein whose rate of penicillin binding ... [more ▼]

Tyrosine-159 of the Streptomyces R61 penicillin-sensitive DD-peptidase was replaced by serine or phenylalanine. The second mutation yielded a very poorly active protein whose rate of penicillin binding was also drastically decreased, except for the reactions with nitrocefin and methicillin. The consequences of the first mutation were more surprising, since a large proportion of the thiolesterase activity was retained, together with the penicillin-binding capacity. Conversely, the peptidase properties was severely affected. In both cases, a drastic decrease in the transferase activity was observed. The results are compared with those obtained by mutation of the corresponding residue in the class A beta-lactamase of Streptomyces albus G. [less ▲]

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See detailA New, Highly Sensitive Method for the Detection and Quantification of Penicillin-Binding Proteins
Galleni, Moreno ULg; Lakaye, Bernard ULg; Lepage, Sophie et al

in Biochemical Journal (1993), 291((Pt 1)), 19-21

A new method for the identification and quantification of penicillin-binding proteins is described which uses fluorescein-coupled penicillins. It allows the rapid detection of 0.2 pmol with the naked eye ... [more ▼]

A new method for the identification and quantification of penicillin-binding proteins is described which uses fluorescein-coupled penicillins. It allows the rapid detection of 0.2 pmol with the naked eye and 2 fmol with the help of an A.L.F. automatic DNA sequencer. Direct labelling can also be performed on whole bacterial cells. [less ▲]

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See detailMechanism of action of DD-peptidases: role of asparagine-161 in the Streptomyces R61 DD-peptidase.
Wilkin, J M; Jamin, M; Joris, Bernard ULg et al

in Biochemical Journal (1993), 293 ( Pt 1)

The role of residue Asn-161 in the interaction between the Streptomyces R61 DD-peptidase and various substrates or beta-lactam inactivators was probed by site-directed mutagenesis. The residue was ... [more ▼]

The role of residue Asn-161 in the interaction between the Streptomyces R61 DD-peptidase and various substrates or beta-lactam inactivators was probed by site-directed mutagenesis. The residue was successively replaced by serine and alanine. In the first case, acylation rates were mainly affected with the peptide and ester substrates but not with the thiol-ester substrates and beta-lactams. However, the deacylation rates were decreased 10-30-fold with the substrates yielding benzoylglycyl and benzoylalanyl adducts. The Asn161Ala mutant was more generally affected, although the acylation rates with cefuroxime and cefotaxime remained similar to those observed with the wild-type enzyme. Surprisingly, the deacylation rates of the benzoylglycyl and benzoylalanyl adducts were very close to those observed with the wild-type enzyme. The results also indicate that the interaction with the peptide substrate and the transpeptidation reaction were more sensitive to the mutations than the other reactions studied. The results are discussed and compared with those obtained with the Asn-132 mutants of a class A beta-lactamase. [less ▲]

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See detailA comparative study of class-D beta-lactamases.
Ledent, Philippe; Raquet, X; Joris, Bernard ULg et al

in Biochemical Journal (1993), 292 ( Pt 2)

Three class-D beta-lactamases (OXA2, OXA1 and PSE2) were produced and purified to protein homogeneity. 6 beta-Iodopenicillanate inactivated the OXA2 enzyme without detectable turnover. Labelling of the ... [more ▼]

Three class-D beta-lactamases (OXA2, OXA1 and PSE2) were produced and purified to protein homogeneity. 6 beta-Iodopenicillanate inactivated the OXA2 enzyme without detectable turnover. Labelling of the same beta-lactamase with 6 beta-iodo[3H]penicillanate allowed the identification of Ser-70 as the active-site serine residue. In agreement with previous reports, the apparent M(r) of the OXA2 enzyme as determined by molecular-sieve filtration, was significantly higher than that deduced from the gene sequence, but this was not due to an equilibrium between a monomer and a dimer. The heterogeneity of the OXA2 beta-lactamase on ion-exchange chromatography contrasted with the similarity of the catalytic properties of the various forms. A first overview of the enzymic properties of the three 'oxacillinases' is presented. With the OXA2 enzyme, 'burst' kinetics, implying branched pathways, seemed to prevail with many substrates. [less ▲]

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See detailAn overview of the kinetic parameters of class B beta-lactamases.
Felici, A; Amicosante, G; Oratore, A et al

in Biochemical Journal (1993), 291 ( Pt 1)

The catalytic properties of three class B beta-lactamases (from Pseudomonas maltophilia, Aeromonas hydrophila and Bacillus cereus) were studied and compared with those of the Bacteroides fragilis enzyme ... [more ▼]

The catalytic properties of three class B beta-lactamases (from Pseudomonas maltophilia, Aeromonas hydrophila and Bacillus cereus) were studied and compared with those of the Bacteroides fragilis enzyme. The A. hydrophila beta-lactamase exhibited a unique specificity profile and could be considered as a rather specific 'carbapenemase'. No relationships were found between sequence similarities and catalytic properties. The problem of the repartition of class B beta-lactamases into sub-classes is discussed. Improved purification methods were devised for the P. maltophilia and A. hydrophila beta-lactamases including, for the latter enzyme, a very efficient affinity chromatography step on a Zn(2+)-chelate column. [less ▲]

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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 thiolester 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 Phe58----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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