References of "Rossolini, G. M"
     in
Bookmark and Share    
Full Text
Peer Reviewed
See detailProbing the specificity of the subclass B3 FEZ-1 metallo-beta-lactamase by site-directed mutagenesis
Mercuri, P. S.; 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 ▲]

Detailed reference viewed: 10 (0 ULg)
Full Text
Peer Reviewed
See detailThe 1.5-angstrom structure of Chryseobacterium meningosepticum zinc beta-lactamase in complex with the inhibitor, D-captopril
Garcia-Saez, I.; Hopkins, J.; Papamicael, C. et al

in Journal of Biological Chemistry (2003), 278(26), 23868-23873

The crystal structure of the class-B beta-lactamase, BlaB, from the pathogenic bacterium, Chryseobacterium meningosepticum, in complex with the inhibitor, D-captopril, has been solved at 1.5-Angstrom ... [more ▼]

The crystal structure of the class-B beta-lactamase, BlaB, from the pathogenic bacterium, Chryseobacterium meningosepticum, in complex with the inhibitor, D-captopril, has been solved at 1.5-Angstrom resolution. The enzyme has the typical alphabeta/betaalpha metallo-beta-lactamase fold and the characteristic two metal binding sites of members of the subclass B1, in which two Zn2+ ions were identified. D-Captopril, a diastereoisomer of the commercial drug, captopril, acts as an inhibitor by displacing the catalytic hydroxyl ion required for antibiotic hydrolysis and intercalating its sulfhydryl group between the two Zn2+ ions. Interestingly, D-captopril is located on one side of the active site cleft. The x-ray structure of the complex of the closely related enzyme, IMP-1, with a mercaptocarboxylate inhibitor, which also contains a sulfhydryl group bound to the two Zn2+ ions, shows the ligand to be located on the opposite side of the active site cleft. A molecule generated by fusion of these two inhibitors would cover the entire cleft, suggesting an interesting approach to the design of highly specific inhibitors. [less ▲]

Detailed reference viewed: 162 (0 ULg)
Full Text
Peer Reviewed
See detailOn functional and structural heterogeneity of VIM-type metallo-beta-lactamases
Docquier, J. D.; Lamotte-Brasseur, J.; Galleni, Moreno ULg et al

in Journal of Antimicrobial Chemotherapy (2003), 51(2), 257-266

The VIM metallo-beta-lactamases are emerging resistance determinants, encoded by mobile genetic elements, that have recently been detected in multidrug-resistant nosocomial isolates of Pseudomonas ... [more ▼]

The VIM metallo-beta-lactamases are emerging resistance determinants, encoded by mobile genetic elements, that have recently been detected in multidrug-resistant nosocomial isolates of Pseudomonas aeruginosa and other Gram-negative pathogens. In this work a T7-based expression system for overproduction of the VIM-2 enzyme by Escherichia coli was developed, which yielded similar to80 mg of protein per litre of culture. The enzyme was mostly released into the medium, from which it was recovered at >99% purity by an initial ammonium sulphate precipitation followed by two chromatography steps, with almost 80% efficiency. Determination of kinetic parameters of VIM-2 under the same experimental conditions previously used for VIM-1 (the first VIM-type enzyme detected in clinical isolates, which is 93% identical to VIM-2) revealed significant differences in K-m values and/or turnover rates with several substrates, including penicillins, cephalosporins and carbapenems. Compared with VIM-1, VIM-2 is more susceptible to inactivation by chelators, indicating that the zinc ions of the latter are probably more loosely bound. These data indicated that at least some of the amino acid differences between the two proteins have functional significance. Molecular modelling of the two enzymes identified some amino acid substitutions, including those at positions 223, 224 and 228 (in the BBL numbering), that could be relevant to the changes in catalytic behaviour. [less ▲]

Detailed reference viewed: 16 (0 ULg)
Full Text
Peer Reviewed
See detailThree-dimensional structure of FEZ-1, a monomeric subclass B3 metallo-beta-lactamase from Fluoribacter gormanii, in native form and in complex with D-captopril
Garcia-Saez, I.; Mercuri, P. S.; Papamicael, C. et al

in Journal of Molecular Biology (2003), 325(4), 651-660

The beta-lactamases are involved in bacterial resistance to penicillin and related compounds. Members of the metallo-enzyme class are now found in many pathogenic bacteria and are thus becoming of major ... [more ▼]

The beta-lactamases are involved in bacterial resistance to penicillin and related compounds. Members of the metallo-enzyme class are now found in many pathogenic bacteria and are thus becoming of major clinical importance. The structures of the Zn-beta-lactamase from Fluoribacter gormanii (FEZ-1) in the native and in the complex form are reported here. FEZ-I is a monomeric enzyme, which possesses two zinc-binding sites. These structures are discussed in comparison with those of the tetrameric L1 enzyme produced by Stenotrophomonas maltophilia. From this analysis, amino acids involved in the oligomerization of L1 are clearly identified. Despite the similarity in fold, the active site of FEZ-1 was found to be significantly different. Two residues, which were previously implicated in function, are not present in L1 or in FEZ-1. The broad-spectrum substrate profile of Zn-beta-lactamases arises from the rather wide active-site cleft, where various P-lactam compounds can be accommodated. (C) 2003 Elsevier Science Ltd. All rights reserved. [less ▲]

Detailed reference viewed: 10 (0 ULg)
Full Text
Peer Reviewed
See detailRole of Cys221 and Asn116 in the zinc-binding sites of the Aeromonas hydrophila metallo-beta-lactamase.
Vanhove, Marc; Zakhem, M.; Devreese, B. et al

in Cellular and molecular life sciences : CMLS (2003), 60(11), 2501-9

The CphA metallo-beta-lactamase produced by Aeromonas hydrophila exhibits two zinc-binding sites. Maximum activity is obtained upon binding of one zinc ion, whereas binding of the second zinc ion results ... [more ▼]

The CphA metallo-beta-lactamase produced by Aeromonas hydrophila exhibits two zinc-binding sites. Maximum activity is obtained upon binding of one zinc ion, whereas binding of the second zinc ion results in a drastic decrease in the hydrolytic activity. In this study, we analyzed the role of Asn116 and Cys221, two residues of the active site. These residues were replaced by site-directed mutagenesis and the different mutants were characterized. The C221S and C221A mutants were seriously impaired in their ability to bind the first, catalytic zinc ion and were nearly completely inactive, indicating a major role for Cys221 in the binding of the catalytic metal ion. By contrast, the binding of the second zinc ion was only slightly affected, at least for the C221S mutant. Mutation of Asn116 did not lead to a drastic decrease in the hydrolytic activity, indicating that this residue does not play a key role in the catalytic mechanism. However, the substitution of Asn116 by a Cys or His residue resulted in an approximately fivefold increase in the affinity for the second, inhibitory zinc ion. Together, these data suggested that the first zinc ion is located in the binding site involving the Cys221 and that the second zinc ion binds in the binding site involving Asn116 and, presumably, His118 and His196. [less ▲]

Detailed reference viewed: 27 (0 ULg)
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 13 (0 ULg)
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 10 (0 ULg)
Full Text
Peer Reviewed
See detailClonal diversity and metallo-beta-lactamase production in clinical isolates of Stenotrophomonas maltophilia
Mercuri, P. S.; 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 ▲]

Detailed reference viewed: 16 (0 ULg)
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 76 (2 ULg)
Full Text
Peer Reviewed
See detailBiochemical Characterization of the Fez-1 Metallo-Beta-Lactamase of Legionella Gormanii Atcc 33297t Produced in Escherichia Coli
Mercuri, P. S.; 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 ▲]

Detailed reference viewed: 20 (1 ULg)
Full Text
Peer Reviewed
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

Detailed reference viewed: 12 (0 ULg)
Full Text
Peer Reviewed
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 ▲]

Detailed reference viewed: 19 (4 ULg)
Full Text
Peer Reviewed
See detailThe Legionella (Fluoribacter) gormanii metallo-beta-lactamase: a new member of the highly divergent lineage of molecular-subclass B3 beta-lactamases.
Boschi, L.; Mercuri, Paola ULg; Riccio, M. L. et al

in Antimicrobial agents and chemotherapy (2000), 44(6), 1538-43

A metallo-beta-lactamase determinant was cloned from a genomic library of Legionella (Fluoribacter) gormanii ATCC 33297(T) constructed in the plasmid vector pACYC184 and transformed into Escherichia coli ... [more ▼]

A metallo-beta-lactamase determinant was cloned from a genomic library of Legionella (Fluoribacter) gormanii ATCC 33297(T) constructed in the plasmid vector pACYC184 and transformed into Escherichia coli DH5alpha, by screening for clones showing a reduced susceptibility to imipenem. The product of the cloned determinant, named FEZ-1, contains a 30-kDa polypeptide and exhibits an isoelectric pH of 7.6. Sequencing revealed that FEZ-1 is a molecular-class B beta-lactamase which shares the closest structural similarity (29.7% of identical residues) with the L1 enzyme of Stenotrophomonas maltophilia, being a new member of the highly divergent subclass B3 lineage. All the residues that in L1 are known to be directly or indirectly involved in coordination of the zinc ions were found to be conserved also in FEZ-1, suggesting that the geometry of zinc coordination in the active site of the latter enzyme is identical to that of L1. Unlike L1, however, FEZ-1 appeared to be monomeric in gel permeation chromatography experiments and exhibited a distinctive substrate specificity with a marked preference for cephalosporins and meropenem. The properties of FEZ-1 overall resembled those of a beta-lactamase previously purified from the same strain of L. gormanii (T. Fujii, K. Sato, K. Miyata, M. Inoue, and S. Mitsuhashi, Antimicrob. Agents Chemother. 29:925-926, 1986) and are as yet unique among class B enzymes, reinforcing the notion that considerable functional heterogeneity can be encountered among members of this class. A system for overexpression of the bla(FEZ-1) gene in E. coli, based on the T7 phage promoter, was also developed. [less ▲]

Detailed reference viewed: 12 (0 ULg)
Full Text
Peer Reviewed
See detailPurification and biochemical characterization of the VIM-1 metallo-beta-lactamase.
Franceschini, N.; Caravelli, B.; Docquier, J. D. et al

in Antimicrobial agents and chemotherapy (2000), 44(11), 3003-7

VIM-1 is a new group 3 metallo-beta-lactamase recently detected in carbapenem-resistant nosocomial isolates of Pseudomonas aeruginosa from the Mediterranean area. In this work, VIM-1 was purified from an ... [more ▼]

VIM-1 is a new group 3 metallo-beta-lactamase recently detected in carbapenem-resistant nosocomial isolates of Pseudomonas aeruginosa from the Mediterranean area. In this work, VIM-1 was purified from an Escherichia coli strain carrying the cloned bla(VIM-1) gene by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. The purified enzyme exhibited a molecular mass of 26 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an acidic pI of 5.1 in analytical isoelectric focusing. Amino-terminal sequencing showed that mature VIM-1 results from the removal of a 26-amino-acid signal peptide from the precursor. VIM-1 hydrolyzes a broad array of beta-lactam compounds, including penicillins, narrow- to expanded-spectrum cephalosporins, carbapenems, and mechanism-based serine-beta-lactamase inactivators. Only monobactams escape hydrolysis. The highest catalytic constant/K(m) ratios (>10(6) M(-1). s(-1)) were observed with carbenicillin, azlocillin, some cephalosporins (cephaloridine, cephalothin, cefuroxime, cefepime, and cefpirome), imipenem, and biapenem. Kinetic parameters showed remarkable variability with different beta-lactams and also within the various penam, cephem, and carbapenem compounds, resulting in no clear preference of the enzyme for any of these beta-lactam subfamilies. Significant differences were observed with some substrates between the kinetic parameters of VIM-1 and those of other metallo-beta-lactamases. Inactivation assays carried out with various chelating agents (EDTA, 1,10-o-phenanthroline, and pyridine-2,6-dicarboxylic acid) indicated that formation of a ternary enzyme-metal-chelator complex precedes metal removal from the zinc center of the protein and revealed notable differences in the inactivation parameters of VIM-1 with different agents. [less ▲]

Detailed reference viewed: 19 (0 ULg)
Full Text
Peer Reviewed
See detailKinetic and spectroscopic characterization of native and metal-substituted beta-lactamase from Aeromonas hydrophila AE036.
Hernandez Valladares, M.; Kiefer, M.; Heinz, U. et al

in FEBS letters (2000), 467(2-3), 221-5

Two metal ion binding sites are conserved in metallo-beta-lactamase from Aeromonas hydrophila. The ligands of a first zinc ion bound with picomolar dissociation constant were identified by EXAFS ... [more ▼]

Two metal ion binding sites are conserved in metallo-beta-lactamase from Aeromonas hydrophila. The ligands of a first zinc ion bound with picomolar dissociation constant were identified by EXAFS spectroscopy as one Cys, two His and one additional N/O donor. Sulfur-to-metal charge transfer bands are observed for all mono- and di-metal species substituted with Cu(II) or Co(II) due to ligation of the single conserved cysteine residue. Binding of a second metal ion results in non-competitive inhibition which might be explained by an alternative kinetic mechanism. A possible partition of metal ions between the two binding sites is discussed. [less ▲]

Detailed reference viewed: 37 (0 ULg)
Full Text
Peer Reviewed
See detailStructure of In31, a Blaimp-Containing Pseudomonas Aeruginosa Integron Phyletically Related to In5, Which Carries an Unusual Array of Gene Cassettes
Laraki, Nadine ULg; Galleni, Moreno ULg; Thamm, Iris ULg et al

in Antimicrobial Agents and Chemotherapy (1999), 43(4), 890-901

The location and environment of the acquired blaIMP gene, which encodes the IMP-1 metallo-beta-lactamase, were investigated in a Japanese Pseudomonas aeruginosa clinical isolate (isolate 101/1477) that ... [more ▼]

The location and environment of the acquired blaIMP gene, which encodes the IMP-1 metallo-beta-lactamase, were investigated in a Japanese Pseudomonas aeruginosa clinical isolate (isolate 101/1477) that produced the enzyme. In this isolate, blaIMP was carried on a 36-kb plasmid, and similar to the identical alleles found in Serratia marcescens and Klebsiella pneumoniae clinical isolates, it was located on a mobile gene cassette inserted into an integron. The entire structure of this integron, named In31, was determined. In31 is a class 1 element belonging to the same group of defective transposon derivatives that originated from Tn402-like ancestors such as In0, In2, and In5. The general structure of In31 appeared to be most closely related to that of In5 from pSCH884, suggesting a recent common phylogeny for these two elements. In In31, the blaIMP cassette is the first of an array of five gene cassettes that also includes an aacA4 cassette and three original cassettes that have never been described in other integrons. The novel cassettes carry, respectively, (i) a new chloramphenicol acetyltransferase-encoding allele of the catB family, (ii) a qac allele encoding a new member of the small multidrug resistance family of proteins, and (iii) an open reading frame encoding a protein of unknown function. All the resistance genes carried on cassettes inserted in In31 were found to be functional in decreasing the in vitro susceptibilities of host strains to the corresponding antimicrobial agents. [less ▲]

Detailed reference viewed: 16 (1 ULg)
Full Text
Peer Reviewed
See detailCloning of a Chryseobacterium (Flavobacterium) meningosepticum chromosomal gene (blaA(CME)) encoding an extended-spectrum class A beta-lactamase related to the Bacteroides cephalosporinases and the VEB-1 and PER beta-lactamases.
Rossolini, G. M.; Franceschini, N.; Lauretti, L. et al

in Antimicrobial agents and chemotherapy (1999), 43(9), 2193-9

In addition to the BlaB metallo-beta-lactamase, Chryseobacterium (Flavobacterium) meningosepticum CCUG 4310 (NCTC 10585) constitutively produces a 31-kDa active-site serine beta-lactamase, named CME-1 ... [more ▼]

In addition to the BlaB metallo-beta-lactamase, Chryseobacterium (Flavobacterium) meningosepticum CCUG 4310 (NCTC 10585) constitutively produces a 31-kDa active-site serine beta-lactamase, named CME-1, with an alkaline isoelectric pH. The blaA(CME) gene that encodes the latter enzyme was isolated from a genomic library constructed in the Escherichia coli plasmid vector pACYC184 by screening for cefuroxime-resistant clones. Sequence analysis revealed that the CME-1 enzyme is a new class A beta-lactamase structurally divergent from the other members of this class, being most closely related to the VEB-1 (also named CEF-1) and PER beta-lactamases and the Bacteroides chromosomal cephalosporinases. The blaA(CME) determinant is located on the chromosome and exhibits features typical of those of C. meningosepticum resident genes. The CME-1 protein was purified from an E. coli strain that overexpresses the cloned gene via a T7-based expression system by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. Kinetic parameters for several substrates were determined. CME-1 is a clavulanic acid-susceptible extended-spectrum beta-lactamase that hydrolyzes most cephalosporins, penicillins, and monobactams but that does not hydrolyze cephamycins and carbapenems. The enzyme exhibits strikingly different kinetic parameters for different classes of beta-lactams, with both K(m) and k(cat) values much higher for cephalosporins than for penicillins and monobactams. However, the variability of both kinetic parameters resulted in overall similar acylation rates (k(cat)/K(m) ratios) for all types of beta-lactam substrates. [less ▲]

Detailed reference viewed: 14 (0 ULg)
Full Text
Peer Reviewed
See detailBiochemical characterization of the Pseudomonas aeruginosa 101/1477 metallo-beta-lactamase IMP-1 produced by Escherichia coli.
Laraki, N.; Franceschini, N.; Rossolini, G. M. et al

in Antimicrobial agents and chemotherapy (1999), 43(4), 902-6

The blaIMP gene coding for the IMP-1 metallo-beta-lactamase produced by a Pseudomonas aeruginosa clinical isolate (isolate 101/1477) was overexpressed via a T7 expression system in Escherichia coli BL21 ... [more ▼]

The blaIMP gene coding for the IMP-1 metallo-beta-lactamase produced by a Pseudomonas aeruginosa clinical isolate (isolate 101/1477) was overexpressed via a T7 expression system in Escherichia coli BL21 (DE3), and its product was purified to homogeneity with a final yield of 35 mg/liter of culture. The structural and functional properties of the enzyme purified from E. coli were identical to those of the enzyme produced by P. aeruginosa. The IMP-1 metallo-beta-lactamase exhibits a broad-spectrum activity profile that includes activity against penicillins, cephalosporins, cephamycins, oxacephamycins, and carbapenems. Only monobactams escape its action. The enzyme activity was inhibited by metal chelators, of which 1,10-o-phenanthroline and dipicolinic acid were the most efficient. Two zinc-binding sites were found. The zinc content of the P. aeruginosa 101/1477 metallo-beta-lactamase was not pH dependent. [less ▲]

Detailed reference viewed: 13 (0 ULg)
Full Text
Peer Reviewed
See detailMono- and binuclear Zn2+-beta-lactamase. Role of the conserved cysteine in the catalytic mechanism.
Paul-Soto, R.; Bauer, R.; Frère, Jean-Marie ULg et al

in The Journal of biological chemistry (1999), 274(19), 13242-9

When expressed by pathogenic bacteria, Zn2+-beta-lactamases induce resistance to most beta-lactam antibiotics. A possible strategy to fight these bacteria would be a combined therapy with non-toxic ... [more ▼]

When expressed by pathogenic bacteria, Zn2+-beta-lactamases induce resistance to most beta-lactam antibiotics. A possible strategy to fight these bacteria would be a combined therapy with non-toxic inhibitors of Zn2+-beta-lactamases together with standard antibiotics. For this purpose, it is important to verify that the inhibitor is effective under all clinical conditions. We have investigated the correlation between the number of zinc ions bound to the Zn2+-beta-lactamase from Bacillus cereus and hydrolysis of benzylpenicillin and nitrocefin for the wild type and a mutant where cysteine 168 is replaced by alanine. It is shown that both the mono-Zn2+ (mononuclear) and di-Zn2+ (binuclear) Zn2+-beta-lactamases are catalytically active but with different kinetic properties. The mono-Zn2+-beta-lactamase requires the conserved cysteine residue for hydrolysis of the beta-lactam ring in contrast to the binuclear enzyme where the cysteine residue is not essential. Substrate affinity is not significantly affected by the mutation for the mononuclear enzyme but is decreased for the binuclear enzyme. These results were derived from kinetic studies on two wild types and the mutant enzyme with benzylpenicillin and nitrocefin as substrates. Thus, targeting drug design to modify this residue might represent an efficient strategy, the more so if it also interferes with the formation of the binuclear enzyme. [less ▲]

Detailed reference viewed: 19 (2 ULg)
Full Text
Peer Reviewed
See detailCharacterization and sequence of the Chryseobacterium (Flavobacterium) meningosepticum carbapenemase: a new molecular class B beta-lactamase showing a broad substrate profile.
Rossolini, G. M.; Franceschini, N.; Riccio, M. L. et al

in The Biochemical journal (1998), 332 ( Pt 1)

The metallo-beta-lactamase produced by Chryseobacterium (formerly Flavobacterium) meningosepticum, which is the flavobacterial species of greatest clinical relevance, was purified and characterized. The ... [more ▼]

The metallo-beta-lactamase produced by Chryseobacterium (formerly Flavobacterium) meningosepticum, which is the flavobacterial species of greatest clinical relevance, was purified and characterized. The enzyme, named BlaB, contains a polypeptide with an apparent Mr of 26000, and has a pI of 8.5. It hydrolyses penicillins, cephalosporins (including cefoxitin), carbapenems and 6-beta-iodopenicillanate, a mechanism-based inactivator of active-site serine beta-lactamases. The enzyme was inhibited by EDTA, 1-10 phenanthroline and pyridine-2,6-dicarboxylic acid, with different inactivation parameters for each chelating agent. The C. meningosepticum blaB gene was cloned and sequenced. According to the G+C content and codon usage, the blaB gene appeared to be endogenous to the species. The BlaB enzyme showed significant sequence similarity to other class B beta-lactamases, being overall more similar to members of subclass B1, which includes the metallo-enzymes of Bacillus cereus (Bc-II) and Bacteroides fragilis (CcrA) and the IMP-1 enzyme found in various microbial species, and more distantly related to the metallo-beta-lactamases of Aeromonas spp. (CphA, CphA2 and ImiS) and of Stenotrophomonas maltophilia (L1). [less ▲]

Detailed reference viewed: 51 (0 ULg)