References of "Frère, Jean-Marie"
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See detailThe inhibition of metallo-beta-lactamase by thioxo-cephalosporin derivatives
Tsang, W. Y.; Dhanda, A.; Schofield, C. J. et al

in Bioorganic & Medicinal Chemistry Letters (2004), 14(7), 1737-1739

The 8-thioxocephalosporins are poor substrates for the B. cereus metallo beta-lactamase (k(cat)/K-m = 61.4 M-1 s(-1)) and act as weak competitive inhibitors (K-i similar to 700 muM). The hydrolysis ... [more ▼]

The 8-thioxocephalosporins are poor substrates for the B. cereus metallo beta-lactamase (k(cat)/K-m = 61.4 M-1 s(-1)) and act as weak competitive inhibitors (K-i similar to 700 muM). The hydrolysis product of thioxocephalosporin, a thioacid, also inhibits the enzyme competitively with a K-i = 96 muM, whereas the cyclic thioxo-piperazinedione, formed by intramolecular aminolysis of thioxo-cephalexin has a K-i of 29 muM. (C) 2004 Elsevier Ltd. All rights reserved. [less ▲]

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See detailEvidence of an intramolecular interaction between the two domains of the BlaR1 penicillin receptor during the signal transduction
Hanique, Sophie; Colombo, Maria-Luigi; Goormaghtigh, Erik et al

in Journal of Biological Chemistry (2004), 279(14), 14264-14272

The BlaR1 protein is a penicillin-sensory transducer involved in the induction of the Bacillus licheniformis beta-lactamase. The amino-terminal domain of the protein exhibits four transmembrane segments ... [more ▼]

The BlaR1 protein is a penicillin-sensory transducer involved in the induction of the Bacillus licheniformis beta-lactamase. The amino-terminal domain of the protein exhibits four transmembrane segments (TM1-TM4) that form a four-alpha-helix bundle embedded in the plasma bilayer. The carboxyl-terminal domain of 250 amino acids (BlaR-CTD) fused at the carboxyl end of TM4 possesses the amino acid sequence signature of penicillin-binding proteins. This membrane topology suggests that BlaR-CTD and the BlaR-amino-terminal domain are responsible for signal reception and signal transduction, respectively. With the use of phage display experiments, we highlight herein an interaction between BlaR-CTD and the extracellular, 63-amino acid L2 loop connecting TM2 and TM3. This interaction does not occur in the presence of penicillin. This result suggests that binding of the antibiotic to BlaR1 might entail the release of the interaction between L2 and BlaR-CTD, causing a motion of the alpha-helix bundle and transfer of the information to the cytoplasm of the cell. In addition, fluorescence spectroscopy, CD, and Fourier transform IR spectroscopy experiments indicate that in contrast to the behavior of the corresponding Staphylococcus aureus protein, the beta-lactam antibiotic does not induce a drastic conformational change in B. licheniformis BlaR-CTD. [less ▲]

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See detailInteractions between penicillin-binding proteins (PBPs) and two novel classes of PBP inhibitors, arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-ones
Zervosen, Astrid ULiege; Lu, Wei-Ping; Chen, Zhouliang et al

in Antimicrobial Agents and Chemotherapy (2004), 48(3), 961-969

Several non-beta-lactam compounds were active against various gram-positive and gram-negative bacterial strains. The MICs of arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-ones were lower ... [more ▼]

Several non-beta-lactam compounds were active against various gram-positive and gram-negative bacterial strains. The MICs of arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-ones were lower than those of ampicillin and cefotaxime for methicillin-resistant Staphylococcus aureus M1339 and vancomycin-resistant Enterococcus faecium EF12. Several compounds were found to inhibit the cell wall synthesis of S. aureus and the last two steps of peptidoglycan biosynthesis catalyzed by ether-treated cells of Escherichia coli or cell wall membrane preparations of Bacillus megaterium. The effects of the arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-one derivatives on E. coli PBP 3 and PBP 5, Streptococcus pneumoniae PBP 2xS (PBP 2x from a penicillin-sensitive strain) and PBP 2xR (PBP 2x from a penicillin-resistant strain), low-affinity PBP 2a of S. aureus, and the Actinomadura sp. strain R39 and Streptomyces sp. strain R61 DD-peptidases were studied. Some of the compounds exhibited inhibitory activities in the 10 to 100 muM concentration range. The inhibition of PBP 2xS by several of them appeared to be noncompetitive. The dissociation constant for the best inhibitor (K-i = 10 muM) was not influenced by the presence of the substrate. [less ▲]

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See detailAcidophilic adaptation of family 11 endo--1,4-xylanases: Modeling and mutational analysis
De Lemos Esteves, Frédéric ULiege; Ruelle, Virginie; Lamotte-Brasseur, Josette ULiege et al

in Protein Science : A Publication of the Protein Society (2004), 13(5), 12091218

Xyl1 from Streptomyces sp. S38 belongs to the low molecular mass family 11 of endo--1,4-xylanases. Its three-dimensional structure has been solved at 2.0 Å and its optimum temperature and pH for enzymatic ... [more ▼]

Xyl1 from Streptomyces sp. S38 belongs to the low molecular mass family 11 of endo--1,4-xylanases. Its three-dimensional structure has been solved at 2.0 Å and its optimum temperature and pH for enzymatic activity are 60°C and 6.0, respectively. Aspergillus kawachii xylanase XynC belongs to the same family but is an acidophilic enzyme with an optimum pH of 2.0. Structural comparison of Xyl1 and XynC showed differences in residues surrounding the two glutamic acid side chains involved in the catalysis that could be responsible for the acidophilic adaptation of XynC. Mutations W20Y, N48D, A134E, and Y193W were introduced by site-directed mutagenesis and combined in multiple mutants. Trp 20 and Tyr 193 are involved in substrate binding. The Y193W mutation inactivated Xyl1 whereas W20Y decreased the optimum pH of Xyl1 to 5.0 and slightly increased its specific activity. The N48D mutation also decreased the optimum pH of Xyl1 by one unit. The A134E substitution did not induce any change, but when combined with N48D, a synergistic effect was observed with a 1.4 unit decrease in the optimum pH. Modeling showed that the orientations of residue 193 and of the fully conserved Arg 131 are different in acidophilic and alkaline xylanases whereas the introduced Tyr 20 probably modifies the pKa of the acid-base catalyst via residue Asn 48. Docking of a substrate analog in the catalytic site highlighted striking differences between Xyl1 and XynC in substrate binding. Hydrophobicity calculations showed a correlation between acidophilic adaptation and a decreased hydrophobicity around the two glutamic acid side chains involved in catalysis. [less ▲]

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See detailThe ybxI gene of Bacillus subtilis 168 encodes a class D beta-lactamase of low activity
Colombo, Maria-Luigi; Hanique, Sophie; Baurin, Stéphane L. et al

in Antimicrobial Agents and Chemotherapy (2004), 48(2), 484-490

The ybxI gene of Bacillus subtilis 168 encodes a preprotein of 267 amino acid residues, including a putative signal peptide of 23 residues. The YbxI primary structure exhibits high similarity scores with ... [more ▼]

The ybxI gene of Bacillus subtilis 168 encodes a preprotein of 267 amino acid residues, including a putative signal peptide of 23 residues. The YbxI primary structure exhibits high similarity scores with two members of the superfamily of the serine penicillin-recognizing enzymes: the class D beta-lactamases and the hydrophilic carboxy-terminal domains of the BlaR and MecR penicillin receptors. To determine the function and the activity of this putative penicillin-recognizing enzyme, we have subcloned the ybxI gene in the pET-26b expression vector. Transformation of Escherichia coli BL21(DE3) by the recombinant plasmid pCIP51 resulted in the export of the mature YbxI in the periplasm as a water-soluble protein. The recombinant protein was purified to 95% homogeneity. YbxI interacts with several beta-lactam antibiotics and can hydrolyze some of them. YbxI is not inactivated by clavulanic acid. The YbxI function and its enzymatic activity in B. subtilis remain unknown. The acyl-enzyme obtained after incubation of YbxI with a fluorescent derivative of ampicillin can be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, confirming that YbxI can be acylated by beta-lactam antibiotics. YbxI does not hydrolyze some of the standard substrates of D-alanyl-D-alanine peptidases, the targets of penicillin. YbxI belongs to the penicillin-recognizing enzyme family but has an activity intermediate between those of a penicillin-binding protein and a beta-lactamase. [less ▲]

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See detailMutational analysis of the catalytic centre of the Citrobacter freundii AmpD N-acetylmuramyl-L-alanine amidase
Genereux, Catherine ULiege; Dehareng, Dominique ULiege; Devreese, Bart et al

in Biochemical Journal (2004), 377(Pt 1), 111-120

Citrobacter freundii AmpD is an intracellular 1,6-anhydro-N-acetylmuramyl-L-alanine amidase involved in both peptidoglycan recycling and beta-lactamase induction. AmpD exhibits a strict specificity for 1 ... [more ▼]

Citrobacter freundii AmpD is an intracellular 1,6-anhydro-N-acetylmuramyl-L-alanine amidase involved in both peptidoglycan recycling and beta-lactamase induction. AmpD exhibits a strict specificity for 1,6-anhydromuropeptides and requires zinc for enzymic activity. The AmpD three-dimensional structure exhibits a fold similar to that of another Zn2+ N-acetylmuramyl-L-alanine amidase, the T7 lysozyme, and these two enzymes define a new family of Zn-amidases which can be related to the eukaryotic PGRP (peptidoglycan-recognition protein) domains. In an attempt to assign the different zinc ligands and to probe the catalytic mechanism of AmpD amidase, molecular modelling based on the NMR structure and site-directed mutagenesis were performed. Mutation of the two residues presumed to act as zinc ligands into alanine (H34A and D164A) yielded inactive proteins which had also lost their ability to bind zinc. By contrast, the active H154N mutant retained the capacity to bind the metal ion. Three other residues which could be involved in the AmpD catalytic mechanism have been mutated (Y63F, E116A, K162H and K162Q). The E116A mutant was inactive, but on the basis of the molecular modelling this residue is not directly involved in the catalytic mechanism, but rather in the binding of the zinc by contributing to the correct orientation of His-34. The K162H and K162Q mutants retained very low activity (0.7 and 0.2% of the wildtype activity respectively), whereas the Y63F mutant showed 16% of the wild-type activity. These three latter mutants exhibited a good affinity for Zn ions and the substituted residues are probably involved in the binding of the substrate. We also describe a new method for generating the N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-tripeptide AmpD substrate from purified peptidoglycan by the combined action of two hydrolytic enzymes. [less ▲]

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See detailB-Lactam Antibiotics
Ghuysen, Jean-Marie ULiege; Frère, Jean-Marie ULiege

in Encyclopedic Reference of Molecular Pharmacology (2004)

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See detailNovel use of lipopeptide preparations
Deleu, Magali ULiege; Brans, Alain; Brasseur, Robert ULiege et al

Patent (2004)

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See detailElectronic and Molecular Properties of an Adsorbed Protein Monolayer Probed by Two-Color Sum-Frequency Generation Spectroscopy
Dreesen, Laurent ULiege; Humbert, C.; Sartenaer, Y. et al

in Langmuir (2004), 20(17), 7201-7207

Two-color sum-frequency generation spectroscopy (2C-SFG) is used to probe the molecular and electronic properties of an adsorbed layer of the green fluorescent protein mutant 2 (GFPmut2) on a platinum ... [more ▼]

Two-color sum-frequency generation spectroscopy (2C-SFG) is used to probe the molecular and electronic properties of an adsorbed layer of the green fluorescent protein mutant 2 (GFPmut2) on a platinum (111) substrate. First, the spectroscopic measurements, performed under different polarization combinations, and atomic force microscopy (AFM) show that the GFPmut2 proteins form a fairly ordered monolayer on the platinum surface. Next, the nonlinear spectroscopic data provide evidence of particular coupling phenomena between the GFPmut2 vibrational and electronic properties. This is revealed by the occurrence of two doubly resonant sum-frequency generation processes for molecules having both their Raman and infrared transition moments in a direction perpendicular to the sample plane. Finally, our 2C-SFG analysis reveals two electronic transitions corresponding to the absorption and fluorescence energy levels which are related to two different GFPmut2 conformations: the B (anionic) and I forms, respectively. Their observation and wavelength positions attest the keeping of the GFPmut2 electronic properties upon adsorption on the metallic surface. [less ▲]

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See detailCrystal structure of the sensor domain of the BlaR penicillin receptor from Bacillus licheniformis
Kerff, Frédéric ULiege; Charlier, Paulette ULiege; Colombo, Maria Louisa et al

in Biochemistry (2003), 42(44), 12835-12843

As in several staphylococci, the synthesis of the Bacillus licheniformis 749/I beta-lactamase is an inducible phenomenon regulated by a signal-transducing membrane protein BlaR. The C-terminal domain of ... [more ▼]

As in several staphylococci, the synthesis of the Bacillus licheniformis 749/I beta-lactamase is an inducible phenomenon regulated by a signal-transducing membrane protein BlaR. The C-terminal domain of this multimodular protein is an extracellular domain which specifically recognizes beta-lactam antibiotics. When it binds a beta-lactam, a signal is transmitted by the transmembrane region to the intracellular loops. In response, the hydrolytic activity of the BlaR large cytoplasmic L3 loop is induced, and a cascade of reactions is generated, leading to the transcription of the beta-lactamase gene. Here, we describe the crystal structure of the extracellular penicillin-receptor domain of BlaR (residues 346-601) at 2.5 Angstrom resolution in order to understand why this domain, whose folding is very similar to that of class D beta-lactamases, behaves as a highly sensitive penicillin-binding protein rather than a beta-lactamase. Two residues of the BlaR C-terminal domain, Thr452 and Thr542, modify the hydrophobic characteristic of the class D beta-lactamase active site. Both residues seem to be in part responsible for the lack of beta-lactamase activity of the BlaR protein due to the stability of the acyl-enzyme. Although further experimental data are needed to fully understand the transmembrane induction process, the comparison of the BlaR sensor domain structure with those of class D beta-lactamase complexes and penicillin-binding proteins provides interesting elements to hypothesize on possible signal transmission mechanisms. [less ▲]

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See detailCrystal structure of extended-spectrum beta-lactamase Toho-1: Insights into the molecular mechanism for catalytic reaction and substrate specificity expansion
Ibuka, A. S.; Ishii, Y.; Galleni, Moreno ULiege et al

in Biochemistry (2003), 42(36), 10634-10643

The crystallographic structure of the class A beta-lactamase Toho-1, an extended-spectrum beta-lactamase with potent activity against expanded-spectrum cephems, has been determined at 1.65 Angstrom ... [more ▼]

The crystallographic structure of the class A beta-lactamase Toho-1, an extended-spectrum beta-lactamase with potent activity against expanded-spectrum cephems, has been determined at 1.65 Angstrom resolution. The result reveals that the Lys73 side chain can adopt two alternative conformations. The predominant conformation of Lys73 is different from that observed in the E166A mutant, indicating that removal of the Glu166 side chain changes the conformation of the Lys73 side chain and thus the interaction between Lys73 and Glu166. The Lys73 side chain would play an important role in proton relay, switching its conformation from one to the other depending on the circumstances. The electron density map also implies possible rotation of Ser237. Comparison of the Toho-1 structure with the structure of other class A beta-lactamases shows that the hydroxyl group of Ser237 is likely to rotate through interaction with the carboxyl group of the substrate. Another peculiarity is the existence of three sulfate ions positioned in or near the substrate-binding cavity. One of these sulfate ions is tightly bound to the active center, while the other two are held by a region of positive charge formed by two arginine residues, Arg274 and Arg276. This positively charged region is speculated to represent a pseudo-binding site of the beta-lactam antibiotics, presumably catching the methoxyimino group of the third-generation cephems prior to proper binding in the substrate-binding cleft for hydrolysis. This high-resolution structure, together with detailed kinetic analysis of Toho-1, provides a new hypothesis for the catalytic mechanism and substrate specificity of Toho-1. [less ▲]

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See detailA detailed kinetic study of Mox-1, a plasmid-encoded class C beta-lactamase
Alba, J.; Bauvois, C.; Ishii, Y. et al

in FEMS Microbiology Letters (2003), 225(2), 183-188

Surveys of beta-lactamases in different parts of the world show an important increase in class C beta-lactamases, thus the study of these enzymes is becoming an important issue. We created an ... [more ▼]

Surveys of beta-lactamases in different parts of the world show an important increase in class C beta-lactamases, thus the study of these enzymes is becoming an important issue. We created an overproduction system for Mox-1, a plasmid class C beta-lactamase, by cloning the gene encoding this enzyme, and placing it under the control of a T7 promoter, using vector pET 28a. The enzyme, purified by ion exchange chromatography, was used to obtain the molecular mass (38 246), the N-terminal sequence (GEASPVDPLRPVV), and p/ (8.9), and to perform a detailed kinetic study. Cephalotin was used as reporter substrate in the case of poor substrates. The kinetic study showed that benzylpenicillin, cephalotin, cefcapene and moxalactam were good substrates for Mox-1 (k(cat)/K-m values > 2.5 x 10(6) M-1 s(-1)). On the other hand, ceftazidime and cefepime were poor substrates for this enzyme (K-m values > 200 muM). Clavulanic acid had no inhibitory effect on Mox-1 (K-m = 30.2 mM), however aztreonam behaved as an inhibitor of Mox-1 (K-i = 2.85 muM). (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. [less ▲]

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See detailOn the substrate specificity of bacterial DD-peptidases: evidence from two series of peptidoglycan-mimetic peptides
Anderson, J. W.; Adediran, S. A.; Charlier, Paulette ULiege et al

in Biochemical Journal (2003), 373(Part 3), 949-955

The reactions between bacterial DD-peptidases and beta-lactam antibiotics have been studied for many years. Less well understood are the interactions between these enzymes and their natural substrates ... [more ▼]

The reactions between bacterial DD-peptidases and beta-lactam antibiotics have been studied for many years. Less well understood are the interactions between these enzymes and their natural substrates, presumably the peptide moieties of peptidoglycan. In general, remarkably little activity has previously been demonstrated in vitro against potential peptide substrates, although in many cases the peptides employed were non-specific and not homologous with the relevant peptidoglycan. In this paper, the specificity of a panel of DD-peptidases against elements of species-specific D-alanyl-D-alanine peptides has been assessed. In two cases, those of soluble, low-molecular-mass DD-peptidases, high activity against the relevant peptides has been demonstrated. In these cases, the high specificity is towards the free N-terminus of the peptidoglycan fragment. With a number of other enzymes, particularly high-molecular-mass DD-peptidases, little or no activity against these peptides was observed. In separate experiments, the reactivity of the enzymes against the central, largely invariant, peptide stem was examined. None of the enzymes surveyed showed high activity against this structural element although weak specificity in the expected direction towards the one structural variable (D-gammaGln versus D-gammaGlu) was observed. The current state of understanding of the activity of these enzymes in vitro is discussed. [less ▲]

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See detailCrystal structure of Enterobacter cloacae 908R class C beta-lactamase bound to iodo-acetamido-phenyl boronic acid, a transition-state analogue
Wouters, J.; Fonze, E.; Vermeire, M. et al

in Cellular and Molecular Life Sciences (2003), 60(8), 1764-1773

The structures of the, class C beta-lactamase from Enterobacter cloacae 908R alone and in complex with a baronic acid transition-state analogue were determined by X-ray crystallography at 2.1 and 2.3 ... [more ▼]

The structures of the, class C beta-lactamase from Enterobacter cloacae 908R alone and in complex with a baronic acid transition-state analogue were determined by X-ray crystallography at 2.1 and 2.3 Angstrom, respectively. The structure of the enzyme resembles those of other class C beta-lactamases. The structure of the. complex with the transition-state analogue, iodo-acetamido-phenyl boronic acid, shows that the inhibitor is covalently, bound to the active-site serine (Ser64). Binding of the inhibitor within the active site is compared with previously determined structures of complexes with other class C enzymes. The structure of the boronic acid adduct indicates ways to improve the affinity of this class of inhibitors. This structure of 908R class C beta-lactamase in complex with a transitionstate analogue provides further insights into the mechanism of action of these hydrolases. [less ▲]

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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 ▲]

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See detailThe kinetic properties of the carboxy terminal domain of the Bacillus licheniformis 749/I BlaR penicillin-receptor shed a new light on the derepression of beta-lactamase synthesis
Duval, Valérie; Swinnen, Marc; Lepage, Sophie et al

in Molecular Microbiology (2003), 48(6), 1553-1564

To study the properties of the BlaR penicillin-receptor involved in the induction of the Bacillus licheniformis beta-lactamase, the water-soluble carboxy terminal domain of the protein (BlaR-CTD) was ... [more ▼]

To study the properties of the BlaR penicillin-receptor involved in the induction of the Bacillus licheniformis beta-lactamase, the water-soluble carboxy terminal domain of the protein (BlaR-CTD) was overproduced in the periplasm of Escherichia coli JM105 and purified to protein homogeneity. Its interactions with various beta-lactam antibiotics were studied. The second-order acylation rate constants k(2)/K' ranged from 0.0017 to more than 1 muM(-1) s(-1) and the deacylation rate constants were lower than 4x10(-5) s(-1) . These values imply a rapid to very rapid formation of a stable acylated adduct. BlaR-CTD is thus one of the most sensitive penicillin-binding proteins presently described. In the light of these results, the kinetics of beta-lactamase induction in Bacillus licheniformis were re-examined. When starting with a rather high cell density, a good beta-lactamase substrate such as benzylpenicillin is too sensitive to beta-lactamase-mediated hydrolysis to allow full induction. By contrast, a poor beta-lactamase substrate (7-aminocephalosporanic acid) can fully derepress beta-lactamase expression under conditions where interference of the antibiotic with cell growth is observed. These results suggest that acylation of the penicillin receptor is a necessary, but not sufficient, condition for full induction. [less ▲]

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See detailDimerization and DNA binding properties of the Bacillus licheniformis 749/I BlaI repressor
Filée, Patrice ULiege; Vreuls, Christelle ULiege; Herman, Raphaël ULiege et al

in Journal of Biological Chemistry (2003), 278(19), 16482-16487

In the absence of penicillin, the beta-lactamase encoding gene blaP of Bacillus licheniformis 749/I is negatively regulated by the transcriptional repressor BlaI. Three palindromic operator regions are ... [more ▼]

In the absence of penicillin, the beta-lactamase encoding gene blaP of Bacillus licheniformis 749/I is negatively regulated by the transcriptional repressor BlaI. Three palindromic operator regions are recognized by BlaI: two in the blaP promoter (OP1 and OP2) and one (OP3) in the promoter of the blaI-blaR1 operon. In this study, the dissociation constant of the purified BlaI dimer was estimated at 25 muM by equilibrium ultracentrifugation. Quantitative Western blot analysis indicates that the intracellular concentrations of BlaI in B. licheniformis 749/I and Bacillus subtilis transformed by a multicopy plasmid harboring the beta-lactamase locus (blaP-blaI-blaR1) were lower than (1.9 muM) or in the same range as (75 muM) the dissociation constant, respectively. This suggests that BlaI is partially dimeric in the cytoplasm of these strains and interacts in vivo with its operators as a preformed dimer. This hypothesis is supported by band shift assays on an operator containing a randomized half-operator sequence. The global dissociation constants of the operator-BlaI dimer complexes were measured by band shift assays and estimated as K-dOP1=1.7+/-0.5 10(-15) M-2, K-dOP2=3.3+/-0.9 10(-15) M-2, and K-dOP3=10.5+/-2.5 10(-15) M-2. The role of the DNA binding properties of BlaI on the beta-lactamase regulation is discussed. [less ▲]

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See detailAnalysis of the importance of the metallo-beta-lactamase active site loop in substrate binding and catalysis
Moali, C.; Anne, C.; Lamotte-Brasseur, J. et al

in Chemistry & Biology (2003), 10(4), 319-329

The role of the mobile loop comprising residues 60-66 in metallo-beta-lactamases has been studied by site-directed mutagenesis, determination of kinetic parameters for six substrates and two inhibitors ... [more ▼]

The role of the mobile loop comprising residues 60-66 in metallo-beta-lactamases has been studied by site-directed mutagenesis, determination of kinetic parameters for six substrates and two inhibitors, pre-steady-state characterization of the interaction with chromogenic nitrocefin, and molecular modeling. The W64A mutation was performed in IMP-1 and BcII (after replacement of the Bcll 60-66 peptide by that of IMP-1) and always resulted in increased K-i and K. and decreased k(cat)/K-m values, an effect reinforced by complete deletion of the loop. k(cat) values were, by contrast, much more diversely affected, indicating that the loop does not systematically favor the best relative positioning of substrate and enzyme catalytic groups. The hydrophobic nature of the ligand is also crucial to strong interactions with the loop, since imipenem was almost insensitive to loop modifications. [less ▲]

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See detailCatalytic mechanism of the Streptomyces K15 DD-transpeptidase/penicillin-binding protein probed by site-directed mutagenesis and structural analysis
Rhazi, Noureddine ULiege; Charlier, Paulette ULiege; Dehareng, Dominique ULiege et al

in Biochemistry (2003), 42(10), 2895-2906

The Streptomyces K15 penicillin-binding DD-transpeptidase is presumed to be involved in peptide cross-linking during bacterial cell wall peptidoglycan assembly. To gain insight into the catalytic ... [more ▼]

The Streptomyces K15 penicillin-binding DD-transpeptidase is presumed to be involved in peptide cross-linking during bacterial cell wall peptidoglycan assembly. To gain insight into the catalytic mechanism, the roles of residues Lys38, Ser96, and Cys98, belonging to the structural elements defining the active site cleft, have been investigated by site-directed mutagenesis, biochemical studies, and X-ray diffraction analysis. The Lys38His and Ser96Ala mutations almost completely abolished the penicillin binding and severely impaired the transpeptidase activities while the geometry of the active site was essentially the same as in the wild-type enzyme. It is proposed that Lys38 acts as the catalytic base that abstracts a proton from the active serine Ser35 during nucleophilic attack and that Ser96 is a key intermediate in the proton transfer from the Ogamma of Ser35 to the substrate leaving group nitrogen. The role of these two residues should be conserved among penicillin-binding proteins containing the Ser-Xaa-Asn/Cys sequence in motif 2. Conversion of Cys98 into Asn decreased the transpeptidase activity and increased hydrolysis of the thiolester substrate and the acylation rate with most beta-lactam antibiotics. Cys98 is proposed to play the same role as Asn in motif 2 of other penicilloyl serine transferases in properly positioning the substrate for the catalytic process. [less ▲]

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See detailOn functional and structural heterogeneity of VIM-type metallo-beta-lactamases
Docquier, J. D.; Lamotte-Brasseur, J.; Galleni, Moreno ULiege 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 ▲]

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