References of "Frère, Jean-Marie"
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See detailMethod for estimation of low outer membrane permeability to beta-lactam antibiotics
Lakaye, Bernard ULg; Dubus, Alice ULg; Joris, Bernard ULg et al

in Antimicrobial Agents and Chemotherapy (2002), 46(9), 2901-2907

The outer membrane of gram-negative bacteria plays a major role in beta-lactam resistance as it slows down antibiotic entry into the periplasm and therefore acts in synergy with beta-lactamases and efflux ... [more ▼]

The outer membrane of gram-negative bacteria plays a major role in beta-lactam resistance as it slows down antibiotic entry into the periplasm and therefore acts in synergy with beta-lactamases and efflux systems. Up to now, the quantitative estimation of low outer membrane permeability by the method of Zimmermann and Rosselet was difficult because of the secreted and cell surface-associated beta-lactamases. The method presented here uses the acylation of a highly sensitive periplasmic penicillin-binding protein (PBP) (BlaR-CTD) to assess the rate of beta-lactam penetration into the periplasm. The method is dedicated to measurement of low permeability and is only valid when the diffusion rate through the outer membrane is rate limiting. Cytoplasmic membrane associated PBPs do not interfere since they are acylated after the very sensitive BlaR-CTD. This method was used to measure the permeability of beta-lactamase-deficient strains of Enterobacter cloacae and Enterobacter aerogenes to benzylpenicillin, ampicillin, carbenicillin, cefotaxime, aztreonam, and cephacetrile. Except for that of cephacetrile, the permeability coefficients were equal to or below 10(-7) cm/s. For cephacetrile, carbenicillin, and benzylpenicillin, the outer membrane of E. cloacae was 20 to 60 times less permeable than that of Escherichia coli, whereas for cefotaxime, aztreonam, and ampicillin it was, respectively, 400, 1,000, and 700 times less permeable. The permeability coefficient for aztreonam is the lowest ever measured (P = 3.2 X 10(-9) cm/s). Using these values, the MICs for a beta-lactamase-overproducing strain of E. cloacae were successfully predicted, demonstrating the validity of the method. [less ▲]

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See detailSubstrate-activated zinc binding of metallo-beta-lactamases - Physiological importance of the mononuclear enzymes
Wommer, S.; Rival, S.; Heinz, U. et al

in Journal of Biological Chemistry (2002), 277(27), 24142-24147

We have investigated the influence of substrate binding on the zinc ion affinity of representatives from the three metallo-g-lactamase subclasses, B1 (BcII from Bacillus cereus and BlaB from ... [more ▼]

We have investigated the influence of substrate binding on the zinc ion affinity of representatives from the three metallo-g-lactamase subclasses, B1 (BcII from Bacillus cereus and BlaB from Chryseobacterium meningosepticum), B2 (CphA from Aeromonas hydrophila), and B3 (L1 from Stenotrophomonas maltophilia). By competition experiments with metal-free apoenzymes and chromophoric zinc chelators or EDTA, we determined the dissociation constants in the absence and presence of substrates. For the formation of the monozine enzymes we determined constants of 1.8, 5.1, 0.007, and 2.6 nm in the absence and 13.6, 1.8, 1.2, and 5.7 pm in the presence of substrates for Bell, BlaB, CphA, and L1, respectively. A second zinc ion binds in the absence (presence) of substrates with considerably higher dissociation constants, namely 1.8 (0.8), 0.007 (0.025), 50 (1.9), and 0.006 (0.12) mum for BcII, BlaB, CphA, and L1, respectively. We have concluded that the apo form might be the prevailing state of most of the metallo-beta-lactamases under physiological conditions in the absence of substrates. Substrate availability induces a spontaneous self-activation due to a drastic decrease of the dissociation constants, resulting in the formation of active mononuclear enzymes already at picomolar free zinc ion concentrations. In the presence of substrates, the binuclear state of the enzymes only exists at unphysiologic high zinc concentrations and might be of no biological relevance. From the competition experiments with EDTA it is further concluded that the reactivation rate does not depend on the pool of free zinc ions but proceeds via the EDTA-Zn(II)-enzyme ternary complexes. [less ▲]

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See detailOverproduction and biochemical characterization of the Chryseobacterium meningosepticum BlaB metallo-beta-lactamase
Vessillier, S.; Docquier, J. D.; Rival, S. et al

in Antimicrobial Agents and Chemotherapy (2002), 46(6), 1921-1927

The BlaB metallo-beta-lactamase of Chryseobacterium meningosepticum CCUG4310 was overproduced in Escherichia coli by means of a T7 promoter-based expression system. The overproducing system, scaled up in ... [more ▼]

The BlaB metallo-beta-lactamase of Chryseobacterium meningosepticum CCUG4310 was overproduced in Escherichia coli by means of a T7 promoter-based expression system. The overproducing system, scaled up in a 15-liter fermentor, yielded approximately 10 mg of BlaB protein per liter, mostly released in the culture supernatant. The enzyme was purified by two ion-exchange chromatographic steps with an overall yield of 66%. Analysis of the kinetic parameters revealed efficient activities (k(cat)/K-m ratios of >10(6) M-1 s(-1)) toward most penam and carbapenem compounds, with the exception of the 6-alpha-methoxypenam derivative temocillin and of biapenem, which were poorer substrates. Hydrolysis of cephalosporins was overall less efficient, with a remarkable variability that was largely due to variable affinities of the BlaB enzyme for different compounds. BlaB was also able to hydrolyze serine-beta-lactamase inhibitors, including beta-iodopenicillanate, sulbactam and, although less efficiently, tazobactam. [less ▲]

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See detailCAU-1, a subclass B3 metallo-beta-lactamase of low substrate affinity encoded by an ortholog present in the Caulobacter crescentus chromosome
Docquier, J. D.; Pantanella, F.; Giuliani, F. et al

in Antimicrobial Agents and Chemotherapy (2002), 46(6), 1823-1830

The sequenced chromosome of Caulobacter crescentus CB15 encodes a hypothetical protein that exhibits significant similarity (30 to 35% identical residues) to metallo-beta-lactamases of subclass B3. An ... [more ▼]

The sequenced chromosome of Caulobacter crescentus CB15 encodes a hypothetical protein that exhibits significant similarity (30 to 35% identical residues) to metallo-beta-lactamases of subclass B3. An allelic variant of this gene (divergent by 3% of its nucleotides) was cloned in Escherichia coli from C crescentus type strain DSM4727. Expression studies confirmed the metallo-p-lactamase activity of its product, CAU-1. The enzyme produced in E. coli was purified by two ion-exchange chromatography steps. CAU-1 contains a 29-kDa polypeptide with an alkaline isoelectric pH (>9), and unlike the L1 enzyme of Stenotrophomonas maltophilia, the native form is monomeric. Kinetic analysis revealed a preferential activity toward penicillins, carbapenems, and narrow-spectrum cephalosporins, while oxyimino cephalosporins were poorly or not hydrolyzed. Affinities for the various beta-lactams were poor overall (K-m values were always >100 muM and often >400 muM). The interaction with divalent ion chelators appeared to occur by a mechanism similar to that prevailing in other members of subclass B3. In C. crescentus, the CAU-1 enzyme is produced independently of beta-lactam exposure and, interestingly, the bla(CAU) determinant is bracketed by three other genes, including two genes encoding enzymes involved in methionine biosynthesis and a gene encoding a putative transcriptional regulator, in an operon-like structure. The CAU-1 enzyme is the first example of a metallo-beta-lactamase in a member of the alpha subdivision of the class Proteobacteria. [less ▲]

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See detailMutational analysis of the two zinc-binding sites of the Bacillus cereus 569/H/9 metallo-beta-lactamase
De Seny, Dominique ULg; Prosperi, Christelle ULg; Bebrone, Carine ULg et al

in Biochemical Journal (2002), 363(Pt 3), 687-696

The metallo-beta-lactamase BcII from Bacillus cereus 569/H/9 possesses a binuclear zinc centre. The mono-zinc form of the enzyme displays an appreciably high activity. although full efficiency is observed ... [more ▼]

The metallo-beta-lactamase BcII from Bacillus cereus 569/H/9 possesses a binuclear zinc centre. The mono-zinc form of the enzyme displays an appreciably high activity. although full efficiency is observed for the di-zinc enzyme. In an attempt to assign the involvement of the different zinc ligands in the catalytic properties of BcII, individual substitutions of selected amino acids were generated. With the exception of His(116) --> Ser (H116S), C221A and C221S, the mono- and di-zinc forms of all the other mutants were poorly active. The activity of H116S decreases by a factor of 10 when compared with the wild type. The catalytic efficiency of C221A and C221S was zinc-dependent. The monozinc forms of these mutants exhibited a low activity, whereas the catalytic efficiency of their respective di-zinc forms was comparable with that of the wild type. Surprisingly, the zinc contents of the mutants and the wild-type Bell were similar. These data suggest that the affinity of the beta-lactamase for the metal was not affected by the substitution of the ligand. The pH-dependence of the H196S catalytic efficiency indicates that the zinc ions participate in the hydrolysis of the beta-lactam ring by acting as a Lewis acid. The zinc ions activate the catalytic water molecule, but also polarize the carbonyl bond of the beta-lactam ring and stabilize the development of a negative charge on the carbonyl oxygen of the tetrahedral reaction intermediate. Our studies also demonstrate that Asn(233) is not directly involved in the interaction with the substrates. [less ▲]

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See detailThe fate of the Blal repressor during the induction of the Bacillus licheniformis BlaP beta-lactamase
Filée, Patrice ULg; Benlafya, Kamal; Delmarcelle, Michaël ULg et al

in Molecular Microbiology (2002), 44(3), 685-694

The induction of the Staphylococcus aureus BlaZ and Bacillus licheniformis 749/l BIaP beta-lactamases by beta-lactam antibiotics occurs according to similar processes. In both bacteria, the products of ... [more ▼]

The induction of the Staphylococcus aureus BlaZ and Bacillus licheniformis 749/l BIaP beta-lactamases by beta-lactam antibiotics occurs according to similar processes. In both bacteria, the products of the blal and blaR1 genes share a high degree of sequence homology and act as repressors and penicillin-sensory transducers respectively. It has been shown in S. aureus that the Blal repressor, which controls the expression of BlaZ negatively, is degraded after the addition of the inducer. In the present study, we followed the fate of Blal during beta-lactamase induction in B. licheniformis 749/l and in a recombinant Bacillus subtilis 168 strain harbouring the pDML995 plasmid, which carries the B. licheniformis blaP, blal and blaR1 genes. In contrast to the situation in B. licheniformis 749/l, beta-lactamase induction in B. subtilis 168/pDML995 was not correlated with the proteolysis of Blal. To exclude molecular variations undetectable by SDS-PAGE, two-dimensional gel electrophoresis was performed with cellular extracts from uninduced or induced B. subtilis 168/pDML995 cells. No variation in the Blal isoelectric point was observed in induced cells, whereas the DNA-binding property was lost. Cross-linking experiments with dithiobis(succimidylpropionate) confirmed that, in uninduced recombinant B. subtilis cells, Blat was present as a homodimer and that this situation was not altered in induced conditions. This latter result is incompatible with a mechanism of inactivation of Blal by proteolysis and suggests that the inactivation of Blal results from a non-covalent modification by a co-activator and that the subsequent proteolysis of Blal might be a secondary phenomenon. In addition to the presence of this co-activator, our results show that the presence of penicillin stress is also required for full induction of beta-lactamase biosynthesis. [less ▲]

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See detailCrystal structures of the Bacillus licheniformis BS3 class A beta-lactamase and of the acyl-enzyme adduct formed with cefoxitin
Fonzé, Evelyne; Vanhove, Mac; Dive, Georges ULg et al

in Biochemistry (2002), 41(6), 1877-1885

The Bacillus licheniformis BS3 beta-lactamase catalyzes the hydrolysis of the beta-lactam ring of penicillins, cephalosporins, and related compounds. The production of beta-lactamases is the most common ... [more ▼]

The Bacillus licheniformis BS3 beta-lactamase catalyzes the hydrolysis of the beta-lactam ring of penicillins, cephalosporins, and related compounds. The production of beta-lactamases is the most common and thoroughly studied cause of antibiotic resistance. Although they escape the hydrolytic activity of the prototypical Staphylococcus aureus beta-lactamase, many cephems are good substrates for a large number of beta-lactamases. However, the introduction of a 7alpha-methoxy substituent, as in cefoxitin, extends their antibacterial spectrum to many cephalosporin-resistant Gram-negative bacteria. The 7alphamethoxy group selectively reduces the hydrolytic action of many beta-lactamases without having a significant effect on the affinity for the target enzymes, the membrane penicillin-binding proteins. We report here the crystallographic structures of the BS3 enzyme and its acyl-enzyme adduct with cefoxitin at 1.7 Angstrom resolution. The comparison of the two structures reveals a covalent acyl-enzyme adduct with perturbed active site geometry, involving a different conformation of the Omega-loop that bears the essential catalytic Glu166 residue. This deformation is induced by the cefoxitin side chain whose position is constrained by the presence of the alpha-methoxy group. The hydrolytic water molecule is also removed from the active site by the 7beta-carbonyl of the acyl intermediate. In light of the interactions and steric hindrances in the active site of the structure of the BS3-cefoxitin acyl-enzyme adduct, the crucial role of the conserved Asn132 residue is confirmed and a better understanding of the kinetic results emerges. [less ▲]

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See detailBeta-lactamases, an old but ever renascent problem
Matagne, André ULg; Galleni, Moreno ULg; Laraki, Nezha et al

in van Broekhoven, A (Ed.) Novel Frontiers in the Production of Compounds for Biomedical Use (2002)

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See detailClonal diversity and metallo-beta-lactamase production in clinical isolates of Stenotrophomonas maltophilia
Mercuri, Paola ULg; Ishii, Y.; Ma, L. et al

in Microbial Drug Resistance : Mechanism, Epidemiology, & Disease (2002), 8(3), 193-200

Stenotrophomonas maltophilia is a nosocomial pathogen with an intrinsic broad-spectrum resistance to beta-lactam compounds and other antibacterial agents. It produces two chromosomal beta-lactamases: a ... [more ▼]

Stenotrophomonas maltophilia is a nosocomial pathogen with an intrinsic broad-spectrum resistance to beta-lactam compounds and other antibacterial agents. It produces two chromosomal beta-lactamases: a clavulanic acid-sensitive class A (L2) and a tetrameric carbapenemase (L1 or BlaS). We screened 40 S. maltophilia multidrug-resistant clinical isolates recovered between 1995 and 1998 in the Varese Hospital (Italy) for the presence of the metallo-beta-lactamase. The isolates were investigated by phenotypic profiling (enzymatic activity and antibiotic resistance pattern) and molecular methods such as PCR and pulsed-field gel electrophoresis (PFGE) to reveal intraspecies diversity. For the tested S. maltophilia strains, we showed that the beta-lactamase production could be induced by the presence of imipenem (50 mug/ml) in the culture media. Addition of 1 mM dipicolinic acid completely inhibited the hydrolysis of imipenem but decreased that nitrocefin in a strain-dependent manner. Full activity of crude extract towards imipenem could be restored by addition of 1 mM ZnCl2. Finally, the gene encoding the carbapenem-hydrolyzing beta-lactamase from S. maltophilia ULA-511 and 39/95, a clinical strain, were isolated and sequenced. These two strains have a different profile of multidrug resistance. The two metallo-beta-lactamases were found to be isologous. The difference of sensitivity of these two strains was associated to the level of production of the metallo-beta-lactamase. [less ▲]

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See detailMetal Ion Binding and Coordination Geometry for Wild Type and Mutants of Metallo-Beta -Lactamase from Bacillus Cereus 569/H/9 (Bcii): A Combined Thermodynamic, Kinetic, and Spectroscopic Approach
De Seny, Dominique ULg; Heinz, U.; Wommer, S. et al

in Journal of Biological Chemistry (2001), 276(48), 45065-78

One high affinity (nm) and one low affinity (microM) macroscopic dissociation constant for the binding of metal ions were found for the wild-type metallo-beta-lactamase from Bacillus cereus as well as six ... [more ▼]

One high affinity (nm) and one low affinity (microM) macroscopic dissociation constant for the binding of metal ions were found for the wild-type metallo-beta-lactamase from Bacillus cereus as well as six single-site mutants in which all ligands in the two metal binding sites were altered. Surprisingly, the mutations did not cause a specific alteration of the affinity of metal ions for the sole modified binding site as determined by extended x-ray absorption fine structure (EXAFS) and perturbed angular correlation of gamma-rays spectroscopy, respectively. Also UV-visible absorption spectra for the mono-cobalt enzymes clearly contain contributions from both metal sites. The observations of the very similar microscopic dissociation constants of both binding sites in contrast to the significantly differing macroscopic dissociation constants inevitably led to the conclusion that binding to the two metal sites exhibits negative cooperativity. The slow association rates for forming the binuclear enzyme determined by stopped-flow fluorescence measurements suggested that fast metal exchange between the two sites for the mononuclear enzyme hinders the binding of a second metal ion. EXAFS spectroscopy of the mono- and di-zinc wild type enzymes and two di-zinc mutants provide a definition of the metal ion environments, which is compared with the available x-ray crystallographic data. [less ▲]

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See detailQuantitative Analysis of the Stabilization by Substrate of Staphylococcus Aureus Pc1 Beta-Lactamase
Lejeune, Annabelle ULg; Vanhove, Marc; Lamotte-Brasseur, Josette et al

in Chemistry & Biology (2001), 8(8), 831-42

BACKGROUND: The stabilization of enzymes in the presence of substrates has been recognized for a long time. Quantitative information regarding this phenomenon is, however, rather scarce since the enzyme ... [more ▼]

BACKGROUND: The stabilization of enzymes in the presence of substrates has been recognized for a long time. Quantitative information regarding this phenomenon is, however, rather scarce since the enzyme destroys the potential stabilizing agent during the course of the experiments. In this work, enzyme unfolding was followed by monitoring the progressive decrease of the rate of substrate utilization by the Staphylococcus aureus PC1 beta-lactamase, at temperatures above the melting point of the enzyme. RESULTS: Enzyme inactivation was directly followed by spectrophotometric measurements. In the presence of substrate concentrations above the K(m) values, significant stabilization was observed with all tested compounds. A combination of unfolding kinetic measurements and enzymatic studies, both under steady-state and non-steady-state regimes, allowed most of the parameters characteristic of the two concurrent phenomena (i.e. substrate hydrolysis and enzyme denaturation) to be evaluated. In addition, molecular modelling studies show a good correlation between the extent of stabilization, and the magnitude of the energies of interaction with the enzyme. CONCLUSIONS: Our analysis indicates that the enzyme is substantially stabilized towards heat-induced denaturation, independently of the relative proportions of non-covalent Henri-Michaelis complex (ES) and acyl-enzyme adduct (ES*). Thus, for those substrates with which the two catalytic intermediates are expected to be significantly populated, both species (ES and ES*) appear to be similarly stabilized. This analysis contributes a new quantitative approach to the problem. [less ▲]

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See detailKinetic Study of Two Novel Enantiomeric Tricyclic Beta-Lactams Which Efficiently Inactivate Class C Beta-Lactamases
Vilar, M.; Galleni, Moreno ULg; Solmajer, T. et al

in Antimicrobial Agents and Chemotherapy (2001), 45(8), 2215-23

A detailed kinetic study of the interaction between two ethylidene derivatives of tricyclic carbapenems, Lek 156 and Lek 157, and representative beta-lactamases and D-alanyl-D-alanine peptidases (DD ... [more ▼]

A detailed kinetic study of the interaction between two ethylidene derivatives of tricyclic carbapenems, Lek 156 and Lek 157, and representative beta-lactamases and D-alanyl-D-alanine peptidases (DD-peptidases) is presented. Both compounds are very efficient inactivators of the Enterobacter cloacae 908R beta-lactamase, which is usually resistant to inhibition. Preliminary experiments indicate that various extended-spectrum class C beta-lactamases (ACT-1, CMY-1, and MIR-1) are also inactivated. With the E. cloacae 908R enzyme, complete inactivation occurs with a second-order rate constant, k(2)/K', of 2 x 10(4) to 4 x 10(4) M(-1) s(-1), and reactivation is very slow, with a half-life of >1 h. Accordingly, Lek 157 significantly decreases the MIC of ampicillin for E. cloacae P99, a constitutive class C beta-lactamase overproducer. With the other serine beta-lactamases tested, the covalent adducts exhibit a wide range of stabilities, with half-lives ranging from long (>4 h with the TEM-1 class A enzyme), to medium (10 to 20 min with the OXA-10 class D enzyme), to short (0.2 to 0.4 s with the NmcA class A beta-lactamase). By contrast, both carbapenems behave as good substrates of the Bacillus cereus metallo-beta-lactamase (class B). The Streptomyces sp. strain R61 and K15 extracellular DD-peptidases exhibit low levels of sensitivity to both compounds. [less ▲]

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See detailInactivation of Aeromonas Hydrophila Metallo-Beta-Lactamase by Cephamycins and Moxalactam
Zervosen, Astrid ULg; Valladares, Maria Hernandez; Devreese, Bart et al

in European Journal of Biochemistry (2001), 268(13), 3840-50

Incubation of moxalactam and cefoxitin with the Aeromonas hydrophila metallo-beta-lactamase CphA leads to enzyme-catalyzed hydrolysis of both compounds and to irreversible inactivation of the enzyme by ... [more ▼]

Incubation of moxalactam and cefoxitin with the Aeromonas hydrophila metallo-beta-lactamase CphA leads to enzyme-catalyzed hydrolysis of both compounds and to irreversible inactivation of the enzyme by the reaction products. As shown by electrospray mass spectrometry, the inactivation of CphA by cefoxitin and moxalactam is accompanied by the formation of stable adducts with mass increases of 445 and 111 Da, respectively. The single thiol group of the inactivated enzyme is no longer titrable, and dithiothreitol treatment of the complexes partially restores the catalytic activity. The mechanism of inactivation by moxalactam was studied in detail. Hydrolysis of moxalactam is followed by elimination of the 3' leaving group (5-mercapto-1-methyltetrazole), which forms a disulfide bond with the cysteine residue of CphA located in the active site. Interestingly, this reaction is catalyzed by cacodylate. [less ▲]

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See detailCenta as a Chromogenic Substrate for Studying Beta-Lactamases
Bebrone, Carine ULg; Moali, C.; Mahy, F. et al

in Antimicrobial Agents and Chemotherapy (2001), 45(6), 1868-71

CENTA, a chromogenic cephalosporin, is readily hydrolyzed by beta-lactamases of all classes except for the Aeromonas hydrophila metalloenzyme. Although it cannot practically be used for the detection of ... [more ▼]

CENTA, a chromogenic cephalosporin, is readily hydrolyzed by beta-lactamases of all classes except for the Aeromonas hydrophila metalloenzyme. Although it cannot practically be used for the detection of beta-lactamase-producing strains on agar plates, it should be quite useful for kinetic studies and the detection of the enzymes in crude extracts and chromatographic fractions. [less ▲]

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See detailBiochemical Characterization of the Fez-1 Metallo-Beta-Lactamase of Legionella Gormanii Atcc 33297t Produced in Escherichia Coli
Mercuri, Paola ULg; Bouillenne, Fabrice ULg; Boschi, L. et al

in Antimicrobial Agents and Chemotherapy (2001), 45(4), 1254-62

The bla(FEZ-1) gene coding for the metallo-beta-lactamase of Legionella (Fluoribacter) gormanii ATCC 33297T was overexpressed via a T7 expression system in Escherichia coli BL21(DE3)(pLysS). The product ... [more ▼]

The bla(FEZ-1) gene coding for the metallo-beta-lactamase of Legionella (Fluoribacter) gormanii ATCC 33297T was overexpressed via a T7 expression system in Escherichia coli BL21(DE3)(pLysS). The product was purified to homogeneity in two steps with a yield of 53%. The FEZ-1 metallo-beta-lactamase exhibited a broad-spectrum activity profile, with a preference for cephalosporins such as cephalothin, cefuroxime, and cefotaxime. Monobactams were not hydrolyzed. The beta-lactamase was inhibited by metal chelators. FEZ-1 is a monomeric enzyme with a molecular mass of 29,440 Da which possesses two zinc-binding sites. Its zinc content did not vary in the pH range of 5 to 9, but the presence of zinc ions modified the catalytic efficiency of the enzyme. A model of the FEZ-1 three-dimensional structure was built. [less ▲]

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See detailStandard Numbering Scheme for Class B Beta-Lactamases
Galleni, Moreno ULg; Lamotte-Brasseur, J.; Rossolini, G. M. et al

in Antimicrobial Agents and Chemotherapy (2001), 45(3), 660-3

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See detailPurification and Characterization of Pbp4a, a New Low-Molecular-Weight Penicillin-Binding Protein from Bacillus subtilis
Duez, Colette ULg; Vanhove, Marc; Gallet, Xavier et al

in Journal of Bacteriology (2001), 183(5), 1595-9

Penicillin-binding protein 4a (PBP4a) from Bacillus subtilis was overproduced and purified to homogeneity. It clearly exhibits DD-carboxypeptidase and thiolesterase activities in vitro. Although highly ... [more ▼]

Penicillin-binding protein 4a (PBP4a) from Bacillus subtilis was overproduced and purified to homogeneity. It clearly exhibits DD-carboxypeptidase and thiolesterase activities in vitro. Although highly isologous to the Actinomadura sp. strain R39 DD-peptidase (B. Granier, C. Duez, S. Lepage, S. Englebert, J. Dusart, O. Dideberg, J. van Beeumen, J. M. Frere, and J. M. Ghuysen, Biochem. J. 282:781-788, 1992), which is rapidly inactivated by many beta-lactams, PBP4a is only moderately sensitive to these compounds. The second-order rate constant (k(2)/K) for the acylation of the essential serine by benzylpenicillin is 300,000 M(-1) s(-1) for the Actinomadura sp. strain R39 peptidase, 1,400 M(-1) s(-1) for B. subtilis PBP4a, and 7,000 M(-1) s(-1) for Escherichia coli PBP4, the third member of this class of PBPs. Cephaloridine, however, efficiently inactivates PBP4a (k(2)/K = 46,000 M(-1) s(-1)). PBP4a is also much more thermostable than the R39 enzyme. [less ▲]

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See detailCharacterization of OXA-29 from Legionella (Fluoribacter) gormanii: molecular class D beta-lactamase with unusual properties.
Franceschini, N.; Boschi, L.; Pollini, S. et al

in Antimicrobial agents and chemotherapy (2001), 45(12), 3509-16

A class D beta-lactamase determinant was isolated from the genome of Legionella (Fluoribacter) gormanii ATCC 33297(T). The enzyme, named OXA-29, is quite divergent from other class D beta-lactamases ... [more ▼]

A class D beta-lactamase determinant was isolated from the genome of Legionella (Fluoribacter) gormanii ATCC 33297(T). The enzyme, named OXA-29, is quite divergent from other class D beta-lactamases, being more similar (33 to 43% amino acid identity) to those of groups III (OXA-1) and IV (OXA-9, OXA-12, OXA-18, and OXA-22) than to other class D enzymes (21 to 24% sequence identity). Phylogenetic analysis confirmed the closer ancestry of OXA-29 with members of the former groups. The OXA-29 enzyme was purified from an Escherichia coli strain overexpressing the gene via a T7-based expression system by a single ion-exchange chromatography step on S-Sepharose. The mature enzyme consists of a 28.5-kDa polypeptide and exhibits an isoelectric pH of >9. Analysis of the kinetic parameters of OXA-29 revealed efficient activity (k(cat)/K(m) ratios of >10(5) M(-1) x s(-1)) for several penam compounds (oxacillin, methicillin, penicillin G, ampicillin, carbenicillin, and piperacillin) and also for cefazolin and nitrocefin. Oxyimino cephalosporins and aztreonam were also hydrolyzed, although less efficiently (k(cat)/K(m) ratios of around 10(3) M(-1) x s(-1)). Carbapenems were neither hydrolyzed nor inhibitory. OXA-29 was inhibited by BRL 42715 (50% inhibitory concentration [IC(50)], 0.44 microM) and by tazobactam (IC(50), 3.2 microM), but not by clavulanate. It was also unusually resistant to chloride ions (IC(50), >100 mM). Unlike OXA-10, OXA-29 was apparently found as a dimer both in diluted solutions and in the presence of EDTA. Its activity was either unaffected or inhibited by divalent cations. OXA-29 is a new class D beta-lactamase that exhibits some unusual properties likely reflecting original structural and mechanistic features. [less ▲]

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See detailCrystallographic analysis of family 11 endo-beta-1,4-xylanase Xyl1 from Streptomyces sp. S38.
Wouters, J.; Georis, J.; Engher, D. et al

in Acta Crystallographica Section D-Biological Crystallography (2001), 57(Pt 12), 1813-9

Family 11 endo-beta-1,4-xylanases degrade xylan, the main constituent of plant hemicelluloses, and have many potential uses in biotechnology. The structure of Xyl1, a family 11 endo-xylanase from ... [more ▼]

Family 11 endo-beta-1,4-xylanases degrade xylan, the main constituent of plant hemicelluloses, and have many potential uses in biotechnology. The structure of Xyl1, a family 11 endo-xylanase from Streptomyces sp. S38, has been solved. The protein crystallized from ammonium sulfate in the trigonal space group P321, with unit-cell parameters a = b = 71.49, c = 130.30 A, gamma = 120.0 degrees. The structure was solved at 2.0 A by X-ray crystallography using the molecular-replacement method and refined to a final R factor of 18.5% (R(free) = 26.9%). Xyl1 has the overall fold characteristic of family 11 xylanases, with two highly twisted beta-sheets defining a long cleft containing the two catalytic residues Glu87 and Glu177. [less ▲]

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See detailBeta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae.
Conrath, K. E.; Lauwereys, M.; Galleni, Moreno ULg et al

in Antimicrobial Agents and Chemotherapy (2001), 45(10), 2807-12

Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy-chain antibodies (VHHs) against enzymes are known to be potent inhibitors. The immunization of dromedaries ... [more ▼]

Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy-chain antibodies (VHHs) against enzymes are known to be potent inhibitors. The immunization of dromedaries with the TEM-1 and BcII beta-lactamases has lead to the isolation of such single-domain antibody fragments specifically recognizing and inhibiting those beta-lactamases. Two VHHs were isolated that inhibit TEM-1 and one BcII inhibiting VHH was identified. All inhibitory VHHs were tight-binding inhibitors. The 50% inhibitory concentrations were determined for all inhibitors and they were all in the same range as the enzyme concentration used in the assay. Addition of the VHHs to the TEM-1 beta-lactamase, expressed on the surface of bacteria, leads to a higher ampicillin sensitivity of the bacteria. This innovative strategy could generate multiple potent inhibitors for all types of beta-lactamases. [less ▲]

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