References of "Joris, Bernard"
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See detailNMR structure of Citrobacter freundii AmpD, comparison with bacteriophage T7 lysozyme and homology with PGRP domains.
Liepinsh, Edvards; Genereux, Catherine ULg; Dehareng, Dominique ULg et al

in Journal of Molecular Biology (2003), 327(4), 833-42

AmpD is a bacterial amidase involved in the recycling of cell-wall fragments in Gram-negative bacteria. Inactivation of AmpD leads to derepression of beta-lactamase expression, presenting a major pathway ... [more ▼]

AmpD is a bacterial amidase involved in the recycling of cell-wall fragments in Gram-negative bacteria. Inactivation of AmpD leads to derepression of beta-lactamase expression, presenting a major pathway for the acquisition of constitutive antibiotic resistance. Here, we report the NMR structure of AmpD from Citrobacter freundii (PDB accession code 1J3G). A deep substrate-binding pocket explains the observed specificity for low molecular mass substrates. The fold is related to that of bacteriophage T7 lysozyme. Both proteins bind zinc at a conserved site and require zinc for amidase activity, although the enzymatic mechanism seems to differ in detail. The structure-based sequence alignment identifies conserved features that are also conserved in the eukaryotic peptidoglycan recognition protein (PGRP) domains, including the zinc-coordination site in several of them. PGRP domains thus belong to the same fold family and, where zinc-binding residues are conserved, may have amidase activity. This hypothesis is supported by the observation that human serum N-acetylmuramyl-L-alanine amidase seems to be identical with a soluble form of human PGRP-L. [less ▲]

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See detailSolution structural study of BlaI: implications for the repression of genes involved in beta-lactam antibiotic resistance.
Melckebeke, Helene Van; Vreuls, Christelle ULg; Gans, Pierre et al

in Journal of Molecular Biology (2003), 333(4), 711-20

beta-Lactamase and penicillin-binding protein PBP2' mediate staphylococcal resistance to beta-lactam antibiotics, which are otherwise highly clinically effective. Two repressors (BlaI and MecI) regulate ... [more ▼]

beta-Lactamase and penicillin-binding protein PBP2' mediate staphylococcal resistance to beta-lactam antibiotics, which are otherwise highly clinically effective. Two repressors (BlaI and MecI) regulate expression of these inducible proteins. Here, we present the first solution structure of the 82 amino acid residue DNA-binding domain of Bacillus licheniformis BlaI which is very similar in primary sequence to the medically significant Staphyloccocal BlaI and MecI proteins. This structure is composed of a compact core of three alpha-helices and a three-stranded beta-sheet typical of the winged helix protein (WHP) family. The protein/DNA complex was studied by NMR chemical shift comparison between the free and complexed forms of BlaI. Residues involved in DNA interaction were identified and a WHP canonical model of interaction with the operators is proposed. In this model, specific contacts occur between the base-pairs of the TACA motif and conserved amino acid residues of the repressor helix H3. These results help toward understanding the repression and induction mechanism of the genes coding for beta-lactamase and PBP2'. [less ▲]

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See detailIsolation and partial characterization of three pregnancy-associated glycoproteins from the ewe placenta
El Amiri, B.; Remy, Benoit; Melo de Sousa, Noelita ULg et al

in Molecular Reproduction and Development (2003), 64(2), 199-206

Pregnancy-associated glycoproteins (PAGs) are synthesized in the outer epithelial layer of the placenta in artiodactyls. In this work, three novel ovine PAGs were isolated from late-pregnancy fetal ... [more ▼]

Pregnancy-associated glycoproteins (PAGs) are synthesized in the outer epithelial layer of the placenta in artiodactyls. In this work, three novel ovine PAGs were isolated from late-pregnancy fetal cotyledons and characterized biochemically. The isolation procedure included acid and ammonium sulfate precipitations and anion and cation exchange chromatographies. The isolated PAGs have different NH2-terminal amino acid sequences (RGSXLTILPLRNMRDIVY, ISRVSXLTIHPLRNIMDML, and RGSNLTIHPLRNIRD) and apparent molecular masses (55, 57, and 59 kDa). Each shows several isoforms with different pl values. The three proteins share high sequence identity with each other and with other ovine, bovine, and caprine PAGs. They have not been described previously. The ovPAG-59 sequence differs from the previously identified ovPAG-4 sequence (determined by DNA cloning and sequencing) at only one position among the 15 N-terminal residues. The newly characterized ovPAGs and the procedure used to isolate them will be helpful in producing new antisera for investigating PAG secretion in pregnant ewes. [less ▲]

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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 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 detailAdvantages and drawbacks of nanospray for studying noncovalent protein-DNA complexes by mass spectrometry
Gabelica, Valérie ULg; Vreuls, Christelle ULg; Filée, Patrice ULg et al

in Rapid Communications in Mass Spectrometry : RCM (2002), 16(18), 1723-1728

The noncovalent complexes between the BlaI protein dimer (wild-type and GM2 mutant) and its double-stranded DNA operator were studied by nanospray mass spectrometry and tandem mass spectrometry (MS/MS ... [more ▼]

The noncovalent complexes between the BlaI protein dimer (wild-type and GM2 mutant) and its double-stranded DNA operator were studied by nanospray mass spectrometry and tandem mass spectrometry (MS/MS). Reproducibility problems in the nanospray single-stage mass spectra are emphasized. The relative intensities depend greatly on the shape of the capillary tip and on the capillary-cone distance. This results in difficulties in assessing the relative stabilities of the complexes simply from MS' spectra of protein-DNA mixtures. Competition experiments using MS/MS are a better approach to determine relative binding affinities. A competition between histidine-tagged BlaIWT (BlaIWTHis) and the GM2 mutant revealed that the two proteins have similar affinities for the DNA operator, and that they co-dimerize to form heterocomplexes. The low sample consumption of nanospray allows MS/MS spectra to be recorded at different collision energies for different charge states with 1 muL of sample. The MS/MS experiments on the dimers reveal that the GM2 dimer is more kinetically stable in the gas phase than the wild-type dimer. The MS/MS experiments on the complexes shows that the two proteins require the same collision energy to dissociate from the complex. This indicates that the rate-limiting step in the monomer loss from the protein-DNA complex arises from the breaking of the protein-DNA interface rather than the protein-protein interface. The dissociation of the protein-DNA complex proceeds by the loss of a highly charged monomer (carrying about two-thirds of the total charge and one-third of the total mass). MS/MS experiments on a heterocomplex also show that the two proteins BlaIWTHis and BlaIGM2 have slightly different charge distributions in the fragments. This emphasizes the need for better understanding the dissociation mechanisms of biomolecular complexes. [less ▲]

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See detailUse of an Alfexpress DNA Sequencer to Analyze Protein-Nucleic Acid Interactions by Band Shift Assay
Filée, Patrice ULg; Delmarcelle, Michaël ULg; Thamm, Iris ULg et al

in BioTechniques (2001), 30(5), 1044-81050-1

Gel retardation analysis, or band shift assay, is technically the simplest method to investigate protein-nucleic acid interactions. In this report, we describe a nonradioactive band shift assay using a ... [more ▼]

Gel retardation analysis, or band shift assay, is technically the simplest method to investigate protein-nucleic acid interactions. In this report, we describe a nonradioactive band shift assay using a fluorescent DNA target and an ALFexpress automatic DNA sequencer in place of the current method that utilizes radioactively end-labeled DNA target and a standard electrophoresis unit. In our study, the dsDNA targets were obtained by annealing two synthetic oligonucleotides or by PCR. In both cases, a molecule of indodicarbocyanine (CY5) was attached at the 5' OH end of one of the two synthetic oligonucleotides, with a ratio of one molecule of fluorescent dye per molecule of dsDNA. To demonstrate the feasibility of this new band shift assay method, the DNA-binding proteins selected as models were the BlaI and AmpR repressors, which are involved in the induction of the Bacillus licheniformis 749/I and Citrobacter freundii beta-lactamases, respectively. The results show that the use of an automatic DNA sequencer allows easy gel retardation analysis and provides a fast, sensitive, and quantitative method. The ALFexpress DNA sequencer has the same limit of detection as a laser fluorescence scanner and can be used instead of a FluorImager or a Molecular Imager. [less ▲]

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See detailCharacterization of two genes encoding the mitochondrial alternative oxidase in Chlamydomonas reinhardtii
Dinant, Monique; Baurain, Denis ULg; Coosemans, Nadine ULg et al

in Current Genetics (2001), 39(2), 101-108

Two cDNA clones (AOX1 and AOX2) and the corresponding genes encoding the alternative oxidases (AOXs) from Chlamydomonas reinhardtii were isolated and sequenced. The cDNAs, AOX1 and AOX2, contained open ... [more ▼]

Two cDNA clones (AOX1 and AOX2) and the corresponding genes encoding the alternative oxidases (AOXs) from Chlamydomonas reinhardtii were isolated and sequenced. The cDNAs, AOX1 and AOX2, contained open reading frames (ORFs) encoding putative proteins of 360 amino acids and 347 amino acids, respectively. For each of the ORFs, a potential mitochondrial-targeting sequence was found in the 5'-end regions. In comparison to AOX enzymes from plants and fungi, the predicted amino acid sequences of the ORFs showed their highest degree of identity with proteins from Aspergillus niger (38.1% and 37.2%) and Ajellomyces capsulatus (37% and 34.9%). Several residues supposed either to be Fe ligands or to be involved in the ubiquinol-binding site were fully conserved in both C. reinhardtii putative AOX proteins. In contrast, a cysteine residue conserved in the sequences of all higher plants and probably involved in the regulation of the enzyme activity was missing both from the AOX1 and AOX2 amino acid sequences and from protein sequences from various other microorganisms. The transcriptional expression of the AOX1 and AOX2 genes in wild-type cells and in mutant cells deficient in mitochondrial complex III activity was also investigated. [less ▲]

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See detailThe Dppa Gene of Bacillus Subtilis Encodes a New D-Aminopeptidase
Cheggour, Abdelatif; Fanuel, Laurence; Duez, Colette ULg et al

in Molecular Microbiology (2000), 38(3), 504-13

Different strains of Bacillus were screened for their ability to hydrolyse D-alanyl-p-nitroanilide. Activity was detected in Bacillus pumilus, Bacillus brevis, Bacillus licheniformis 749I and Bacillus ... [more ▼]

Different strains of Bacillus were screened for their ability to hydrolyse D-alanyl-p-nitroanilide. Activity was detected in Bacillus pumilus, Bacillus brevis, Bacillus licheniformis 749I and Bacillus subtilis 168. The last strain was the best producer and was selected for the production and purification of the enzyme. The determination of the N-terminal sequence identified the enzyme as the product of the dppA gene (previously named dciAA) belonging to the dipeptide ABC transport (dpp) operon expressed early during sporulation. Open reading frames (ORFs) encoding putative related proteins were found in the genomes of a variety of Archaea and both sporulating and non-sporulating bacteria. The enzyme behaves as a D-aminopeptidase and represents the prototype of a new peptidase family. Among the tested substrates, the highest activities were found with D-Ala-D-Ala and D-Ala-Gly-Gly. The active enzyme behaves as an octamer of identical 30 kDa subunits. It exhibits a broad pH optimum, extending between pH 9 and 11. It is reversibly inhibited in the presence of Zn2+ chelators, and the sequence comparisons highlight the conservation of potential Zn-binding residues. As it has been shown by others that null mutations in the dpp operon do not inhibit spore formation, the physiological role of DppA is probably an adaptation to nutrient deficiency. [less ▲]

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See detailA new variant of the Ntn hydrolase fold revealed by the crystal structure of L-aminopeptidase D-ala-esterase/amidase from Ochrobactrum anthropi.
Bompard-Gilles, C; Villeret, V; Davies, G J et al

in Structure (2000), 8(2), 153-62

BACKGROUND: The L-aminopeptidase D-Ala-esterase/amidase from Ochrobactrum anthropi (DmpA) releases the N-terminal L and/or D-Ala residues from peptide substrates. This is the only known enzyme to liberate ... [more ▼]

BACKGROUND: The L-aminopeptidase D-Ala-esterase/amidase from Ochrobactrum anthropi (DmpA) releases the N-terminal L and/or D-Ala residues from peptide substrates. This is the only known enzyme to liberate N-terminal amino acids with both D and L stereospecificity. The DmpA active form is an alphabeta heterodimer, which results from a putative autocatalytic cleavage of an inactive precursor polypeptide. RESULTS: The crystal structure of the enzyme has been determined to 1.82 A resolution using the multiple isomorphous replacement method. The heterodimer folds into a single domain organised as an alphabetabetaalpha sandwich in which two mixed beta sheets are flanked on both sides by two alpha helices. CONCLUSIONS: DmpA shows no similarity to other known aminopeptidases in either fold or catalytic mechanism, and thus represents the first example of a novel family of aminopeptidases. The protein fold of DmpA does, however, show structural homology to members of the N-terminal nucleophile (Ntn) hydrolase superfamily. DmpA presents functionally equivalent residues in the catalytic centre when compared with other Ntn hydrolases, and is therefore likely to use the same catalytic mechanism. In spite of this homology, the direction and connectivity of the secondary structure elements differ significantly from the consensus Ntn hydrolase topology. The DmpA structure thus characterises a new subfamily, but supports the common catalytic mechanism for these enzymes suggesting an evolutionary relationship. [less ▲]

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See detailTechnique for a rapid and efficient purification of the SHV-1 and PSE-2 beta-lactamases.
Bouillenne, Fabrice ULg; Matagne, André ULg; Joris, Bernard ULg et al

in Journal of Chromatography. B : Biomedical Sciences and Applications (2000), 737(1-2), 261-5

A simple procedure is described which results in an optimised resolution in molecular sieve chromatography. A sample exhibiting a large initial volume (about 20 ml) and conditioned in a buffer of low ... [more ▼]

A simple procedure is described which results in an optimised resolution in molecular sieve chromatography. A sample exhibiting a large initial volume (about 20 ml) and conditioned in a buffer of low ionic strength (<20 mM) by filtration through a 53-ml G25 molecular sieve column, is adsorbed on a 1.7-ml ion-exchange (SOURCE) column. The proteins are released by a 10-ml pulse of 1 M NaCl and the eluate directly injected onto a 120-ml Sephacryl S100-HR column. The very low volume of the eluate ensures optimal conditions and resolution for the molecular sieving process. The method is applied as the polishing step in the purification of the SHV-1 and PSE-2 beta-lactamases. It could easily be scaled up for the treatment of larger samples. [less ▲]

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See detailCrystal structure of a D-aminopeptidase from Ochrobactrum anthropi, a new member of the 'penicillin-recognizing enzyme' family.
Bompard-Gilles, C.; Remaut, H.; Villeret, V. et al

in Structure (2000), 8(9), 971-80

BACKGROUND: beta-Lactam compounds are the most widely used antibiotics. They inactivate bacterial DD-transpeptidases, also called penicillin-binding proteins (PBPs), involved in cell-wall biosynthesis ... [more ▼]

BACKGROUND: beta-Lactam compounds are the most widely used antibiotics. They inactivate bacterial DD-transpeptidases, also called penicillin-binding proteins (PBPs), involved in cell-wall biosynthesis. The most common bacterial resistance mechanism against beta-lactam compounds is the synthesis of beta-lactamases that hydrolyse beta-lactam rings. These enzymes are believed to have evolved from cell-wall DD-peptidases. Understanding the biochemical and mechanistic features of the beta-lactam targets is crucial because of the increasing number of resistant bacteria. DAP is a D-aminopeptidase produced by Ochrobactrum anthropi. It is inhibited by various beta-lactam compounds and shares approximately 25% sequence identity with the R61 DD-carboxypeptidase and the class C beta-lactamases. RESULTS: The crystal structure of DAP has been determined to 1.9 A resolution using the multiple isomorphous replacement (MIR) method. The enzyme folds into three domains, A, B and C. Domain A, which contains conserved catalytic residues, has the classical fold of serine beta-lactamases, whereas domains B and C are both antiparallel eight-stranded beta barrels. A loop of domain C protrudes into the substrate-binding site of the enzyme. CONCLUSIONS: Comparison of the biochemical properties and the structure of DAP with PBPs and serine beta-lactamases shows that although the catalytic site of the enzyme is very similar to that of beta-lactamases, its substrate and inhibitor specificity rests on residues of domain C. DAP is a new member of the family of penicillin-recognizing proteins (PRPs) and, at the present time, its enzymatic specificity is clearly unique. [less ▲]

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See detailThe DmpA aminopeptidase from Ochrobactrum anthropi LMG7991 is the prototype of a new terminal nucleophile hydrolase family.
Fanuel, L; Goffin, Colette ULg; Cheggour, A et al

in Biochemical Journal (1999), 341(Pt 1), 147-55

The DmpA (d-aminopeptidase A) protein produced by Ochrobactrum anthropi hydrolyses p-nitroanilide derivatives of glycine and d-alanine more efficiently than that of l-alanine. When regular peptides are ... [more ▼]

The DmpA (d-aminopeptidase A) protein produced by Ochrobactrum anthropi hydrolyses p-nitroanilide derivatives of glycine and d-alanine more efficiently than that of l-alanine. When regular peptides are utilized as substrates, the enzyme behaves as an aminopeptidase with a preference for N-terminal residues in an l configuration, thus exemplifying an interesting case of stereospecificity reversal. The best-hydrolysed substrate is l-Ala-Gly-Gly, but tetra- and penta-peptides are also efficiently hydrolysed. The gene encodes a 375-residue precursor, but the active enzyme contains two polypeptides corresponding to residues 2-249 (alpha-subunit) and 250-375 (beta-subunit) of the precursor. Residues 249 and 250 are a Gly and a Ser respectively, and various substitutions performed by site-directed mutagenesis result in the production of an uncleaved and inactive protein. The N-terminal Ser residue of the beta-subunit is followed by a hydrophobic peptide, which is predicted to form a beta-strand structure. All these properties strongly suggest that DmpA is an N-terminal amidohydrolase. An exploration of the databases highlights the presence of a number of open reading frames encoding related proteins in various bacterial genomes. Thus DmpA is very probably the prototype of an original family of N-terminal hydrolases. [less ▲]

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See detailTwo new aminopeptidases from Ochrobactrum anthropi active on D-alanyl-p-nitroanilide.
Fanuel, L; Thamm, Iris ULg; Kostanjevecki, V et al

in Cellular and Molecular Life Sciences : CMLS (1999), 55(5), 812-8

Two new enzymes which hydrolyse D-alanyl-p-nitroanilide have been detected in Ochrobactrum anthropi LMG7991 extracts. The first enzyme, DmpB, was purified to homogeneity and found to be homologous to the ... [more ▼]

Two new enzymes which hydrolyse D-alanyl-p-nitroanilide have been detected in Ochrobactrum anthropi LMG7991 extracts. The first enzyme, DmpB, was purified to homogeneity and found to be homologous to the Dap protein produced by O. anthropi SCRC C1-38 (ATCC49237). The second enzyme, DmpA, exhibits a similar substrate profile when tested on p-nitroanilide derivatives of glycine and L/D-alanine, but the amounts produced by the Ochrobactrum strain were not sufficient to allow complete purification. Interestingly, the DmpA preparation also exhibited an L-aminopeptidase activity on the tripeptide L-Ala-Gly-Gly but it was not possible to be certain that the same protein was responsible for both p-nitroanilide and peptide hydrolysing activities. The gene encoding the DmpA protein was cloned and sequenced. The deduced protein sequence exhibits varying degrees of similarity with those corresponding to several open reading frames found in the genomes of other prokaryotic organisms, including Mycobacteria. None of these gene products has been isolated or characterised, but a tentative relationship can be proposed with the NylC amidase from Flavobacterium sp. K172. [less ▲]

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See detailCrystallization and preliminary X-ray analysis of a new L-aminopeptidase-D-amidase/D-esterase activated by a Gly-Ser peptide bond hydrolysis.
Bompard-Gilles, C; Villeret, V; Fanuel, L et al

in Acta Crystallographica Section D-Biological Crystallography (1999), 55(Pt 3), 699-701

Ochrobactrum anthropi possesses an L-aminopeptidase (DmpA) also able to act as a D-amidase/D-esterase. DmpA (40 kDa) is activated by auto-catalyzed protein splicing liberating an alpha-amino group ... [more ▼]

Ochrobactrum anthropi possesses an L-aminopeptidase (DmpA) also able to act as a D-amidase/D-esterase. DmpA (40 kDa) is activated by auto-catalyzed protein splicing liberating an alpha-amino group presumably used as a general base in the catalytic mechanism. Two crystal forms were obtained at 294 K in 13-16% PEG 2000 mono-methylether at pH 9.0, adding either 0.2 M magnesium chloride or 1 M lithium chloride. Crystals of the first form belong to the space group C2221 and diffract to 3.0 A resolution, whereas crystals of the second form belong to the space group P21212 and diffract to 2.3 A resolution. Initial screening for heavy-atom derivatives on form II crystals, has led to a well substituted Hg derivative. [less ▲]

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See detailThe diversity, structure and regulation of beta-lactamases.
Philippon, A; Dusart, Jean; Joris, Bernard ULg et al

in Cellular and Molecular Life Sciences : CMLS (1998), 54(4), 341-6

beta-Lactamase production is responsible for the appearance of a large number of pathogenic bacterial strains exhibiting a high degree of resistance to beta-lactam antibiotics. A large number of enzymes ... [more ▼]

beta-Lactamase production is responsible for the appearance of a large number of pathogenic bacterial strains exhibiting a high degree of resistance to beta-lactam antibiotics. A large number of enzymes have been described with very diverse primary structures and catalytic profiles. Nevertheless, all known three-dimensional structures of active-site serine beta-lactamases exhibit a high degree of similarity with apparently equivalent chemical functionalities in the same strategic positions. These groups might not, however, play identical roles in the various classes of enzymes. Structural data have also been recently obtained for the zinc metallo-beta-lactamases, but the detailed catalytic mechanisms might also differ widely, depending on the enzyme studied. Similarly, the induction of the synthesis of beta-lactamases is now better understood, but many questions remain to be answered. [less ▲]

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See detailRésistance bactérienne aux beta-lactamines
Charlier, Paulette ULg; Coyette, Jacques ULg; Dehareng, Dominique ULg et al

in Medecine Sciences : M/S (1998), 14(5), 544-555

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See detailDual Multimodular Class a Penicillin-Binding Proteins in Mycobacterium Leprae
Lepage, Sophie; Dubois, Philippe ULg; Ghosh, Thushar-Kanti et al

in Journal of Bacteriology (1997), 179(14), 4627-4630

The ponA gene of cosmid L222 of the Mycobacterium leprae genome library encodes a multimodular class A penicillin-binding protein (PBP), PBP1. The PBP, labelled with a polyhistidine sequence, has been ... [more ▼]

The ponA gene of cosmid L222 of the Mycobacterium leprae genome library encodes a multimodular class A penicillin-binding protein (PBP), PBP1. The PBP, labelled with a polyhistidine sequence, has been produced in Escherichia coli, extracted from the membranes with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) and purified by Ni2(+)-nitrilotriacetic acid-agarose chromatography. In contrast to the pon1-encoded class A PBP1, PBP1 undergoes denaturation at temperatures higher than 25 degrees C, it catalyzes acyl transfer reactions on properly structured thiolesters, and it binds penicillin with high affinity. [less ▲]

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See detailUse of an Automatic DNA Sequencer for S1 Mapping: Transcriptional Analysis of the Streptomyces Coelicolor A3(2) Dnak Operon
Brans, Alain ULg; Loriaux, Axelle; Thamm, Iris ULg et al

in FEMS Microbiology Letters (1997), 149(2), 189-94

The transcription start point of the dnaK operon of Streptomyces coelicolor A3(2) has been determined by S1 mapping, using the EMBL automated fluorescent DNA sequencer. The -35 and -10 hexamers correspond ... [more ▼]

The transcription start point of the dnaK operon of Streptomyces coelicolor A3(2) has been determined by S1 mapping, using the EMBL automated fluorescent DNA sequencer. The -35 and -10 hexamers correspond to a sigma 70-type promoter. This promoter responds to heat shock and involves an inverted repeat different from the CIRCE sequence characteristic of the Gram-positive heat-shock promoters. [less ▲]

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See detailPenicillin-binding proteins. Wall peptidoglycan assembly and resistance to penicillin: facts, doubts and hopes
Ghuysen, Jean-Marie ULg; Charlier, Paulette ULg; Coyette, Jacques ULg et al

in International Journal of Antimicrobial Agents (1997), 8(1), 45-60

As the protein sequence and structure databases expand, the relationships between proteins, the notion of protein superfamily, and the driving forces of evolution are better understood. Key steps of the ... [more ▼]

As the protein sequence and structure databases expand, the relationships between proteins, the notion of protein superfamily, and the driving forces of evolution are better understood. Key steps of the synthesis of the bacterial cell wall peptidoglycan are revisited in light of these advances. The reactions through which the D-alanyl-D-alanine depeptide is formed, utilized, and hydrolyzed and the sites of action of the glycopeptide and β-lactam antibiotics illustrate the concept according to which new enzyme functions evolve as a result of tinkering of existing proteins. This occurs by the acquisition of local structural changes, the fusion into mul-timodular polypeptides, and the association into multiprotein complexes. [less ▲]

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