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See detailStudies of the Domains II and III of Bacillus subtilis PBP4a in relation with the protein localization
Vanden Broeck, Arnaud ULg; Van Der Heiden, Edwige ULg; Sauvage, Eric ULg et al

Poster (2014, April 23)

Bacillus subtilis PBP4a belongs to the class-C1 PBPs characterized by two internal additional domains of unknown function. Seven lysine residues (K) are protruding from domain II. Four of them have been ... [more ▼]

Bacillus subtilis PBP4a belongs to the class-C1 PBPs characterized by two internal additional domains of unknown function. Seven lysine residues (K) are protruding from domain II. Four of them have been mutated in glutamine residues (Q). Both proteins (WT and Mut4KQ PBP4a) have been produced without signal peptide in E. coli and their sub-cellular localizations determined by measuring the DD-carboxypeptidase activities in the different compartments (cytoplasmic vs membrane attached proteins). In order to detect a possible influence of the PBP4a domain III in the localization of the protein, its encoding sequence has been cloned into pET-28b-BlaP, a vector allowing the production of WT BlaP β-lactamase or BlaP/DIII chimeric protein (with domain III inserted in a permissive loop of BlaP). The nitrocefin hydrolysis activities of BlaP or BlaP/DIII have been measured in the different cellular compartments. [less ▲]

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See detailStudy of the Bacillus subtilis ATCC21332 pbpE-racX operon in relation with the formation or disassembly of biofilms
Vanden Broeck, Arnaud ULg; Van Der Heiden, Edwige ULg; Joris, Bernard ULg et al

Poster (2013, December 20)

Bacillus subtilis is a PGPR (Plant Growth Promoting Rhizobacterium) Gram positive bacterium and a model for studying the in vitro formation or disruption of biofilms. At the liquid/air interface of ... [more ▼]

Bacillus subtilis is a PGPR (Plant Growth Promoting Rhizobacterium) Gram positive bacterium and a model for studying the in vitro formation or disruption of biofilms. At the liquid/air interface of standing cultures, B. subtilis forms thick pellicles of limited lifetimes. Some D-amino acids have been reported among the factors playing a role in the disassembly of B. subtilis biofilms and ylmE or racX mutants (in which the racemases YlmE or RacX are absent) show a delay in pellicle disruption [I. Kolodkin et al. Science (2010) 328:627-629]. The racX encoding gene is part of a bicistronic operon in which the first gene (pbpE) codes for a Penicillin-Binding Protein, the PBP4* whose function is not characterized. Our studies aim to delete the complete pbpE-racX operon and compare the phenotypes of mutants and parental strains ATCC21332 or ATCC6051 in standing cultures. The substrate specificity of the purified RacX racemase is currently under investigation as well as the functional characterization of PBP4*, a protein possessing a lipocalin-like domain. [less ▲]

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See detailSwitching to the Dark Side: Repositioning of Polyglutamine Repeat Promotes Amyloid Fibril Formation by the Model Protein, β-Lactamase BlaP
Thorn, David ULg; Pain, Coralie ULg; Scarafone, Natacha et al

Conference (2013, November 20)

Background: The expansion of polyglutamine (polyQ) repeats is associated with an increased propensity of the protein to aggregate into amyloid fibrils. There are ten human proteins presently known within ... [more ▼]

Background: The expansion of polyglutamine (polyQ) repeats is associated with an increased propensity of the protein to aggregate into amyloid fibrils. There are ten human proteins presently known within which polyQ expansion above a threshold length, e.g. 35-50 residues, leads to ten distinct neurodegenerative disorders [1], the most well-known being Huntington’s disease. While repeat length, aggregation, and disease are well correlated, recent studies suggest the non-polyQ regions of these proteins can also play a significant role, both preventative and facilitative, in the aggregation process. With the aim of exploring this role in more detail, we have engineered chimeric proteins via the insertion of polyQ repeats of various length (23, 30, 55, 79 Q) into two sites of antibiotic resistance enzyme BlaP β-lactamase from Bacillus licheniformis 749/C [2]. Questions addressed: How does polyQ repeat position affect the structure, stability and aggregation of polyQ proteins? Methods: Aggregation kinetics determined by monitoring the decrease in soluble protein fraction over time. Aggregate morphology examined by transmission electron microscopy. Protein stability derived from thermal or chemical unfolding transitions monitored by far-UV CD and intrinsic fluorescence. Results and discussion: PolyQ insertion at either of the two positions led to a decrease in thermodynamic stability that was largely independent of polyQ length. Chimeras with polyQ insertions at position 216 were destabilised to a much greater extent than those with insertions at position 197. The reduced stability of the 216 chimeras was associated with an increased aggregation propensity: (i) the minimum polyQ length leading to aggregation was lower, and (ii) the aggregation rate was significantly higher than that observed by 197 chimeras with equivalent polyQ lengths. Remarkably, the two sites of polyQ insertion are indeed very similar, both residing within flexible loop regions between stable α-helices. Moreover, the 216 chimeras exhibited a higher aggregation propensity than their 197 counterparts even under denaturing conditions, suggesting the disparity between the two chimeras cannot be accounted for by structural differences alone. These findings highlight the strong and complex influence of the overall protein context on polyQ-mediated aggregation. The molecular basis for the observed changes in stability and aggregation propensity is the subject of on-going work. [less ▲]

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See detailInsertion of domain III of Bacillus subtilis and Bacillus amyloliquefaciens PBP4a in the Bacillus licheniformis BlaP β-lactamase to study the binding to peptidoglycan and whole bacteria
Van Der Heiden, Edwige ULg; Hoebreck, Charline ULg; Freichels, Régine ULg et al

Poster (2013, October 03)

Domain III of Bacillus subtilis and B. amyloliquefaciens DD-endopeptidase PBP4a was introduced in the BlaP beta-lactamse of Bacillus licheniformis. Domain III of Bacillus licheniformis binds to ... [more ▼]

Domain III of Bacillus subtilis and B. amyloliquefaciens DD-endopeptidase PBP4a was introduced in the BlaP beta-lactamse of Bacillus licheniformis. Domain III of Bacillus licheniformis binds to peptidoglycan of Bacillus subtilis 168 and to itself whole cells. [less ▲]

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See detailInhibition of dd-Peptidases by a Specific Trifluoroketone: Crystal Structure of a Complex with the Actinomadura R39 dd-Peptidase.
Dzhekieva, Liudmila; Adediran, S. A.; Herman, Raphael et al

in Biochemistry (2013)

Inhibitors of bacterial dd-peptidases represent potential antibiotics. In the search for alternatives to beta-lactams, we have investigated a series of compounds designed to generate transition state ... [more ▼]

Inhibitors of bacterial dd-peptidases represent potential antibiotics. In the search for alternatives to beta-lactams, we have investigated a series of compounds designed to generate transition state analogue structures upon reaction with dd-peptidases. The compounds contain a combination of a peptidoglycan-mimetic specificity handle and a warhead capable of delivering a tetrahedral anion to the enzyme active site. The latter includes a boronic acid, two alcohols, an aldehyde, and a trifluoroketone. The compounds were tested against two low-molecular mass class C dd-peptidases. As expected from previous observations, the boronic acid was a potent inhibitor, but rather unexpectedly from precedent, the trifluoroketone [d-alpha-aminopimelyl(1,1,1-trifluoro-3-amino)butan-2-one] was also very effective. Taking into account competing hydration, we found the trifluoroketone was the strongest inhibitor of the Actinomadura R39 dd-peptidase, with a subnanomolar (free ketone) inhibition constant. A crystal structure of the complex between the trifluoroketone and the R39 enzyme showed that a tetrahedral adduct had indeed formed with the active site serine nucleophile. The trifluoroketone moiety, therefore, should be considered along with boronic acids and phosphonates as a warhead that can be incorporated into new and effective dd-peptidase inhibitors and therefore, perhaps, antibiotics. [less ▲]

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See detailSubstrate Specificity of Low-Molecular Mass Bacterial DD-Peptidases
Nemmara, Venkatesh V; Dzhekieva, Liudmila; Subarno Sakar, Kumar et al

in Biochemistry (2011), 50

The bacterial DD-peptidases or penicillin-binding proteins (PBPs) catalyze the formation and regulation of cross-links in peptidoglycan biosynthesis. They are classified into two groups, the high ... [more ▼]

The bacterial DD-peptidases or penicillin-binding proteins (PBPs) catalyze the formation and regulation of cross-links in peptidoglycan biosynthesis. They are classified into two groups, the high-molecular mass (HMM) and lowmolecular mass (LMM) enzymes. The latter group, which is subdivided into classes A−C (LMMA, -B, and -C, respectively), is believed to catalyze DD-carboxypeptidase and endopeptidase reactions in vivo. To date, the specificity of their reactions with particular elements of peptidoglycan structure has not, in general, been defined. This paper describes the steady-state kinetics of hydrolysis of a series of specific peptidoglycan-mimetic peptides, representing various elements of stem peptide structure, catalyzed by a range of LMM PBPs (the LMMA enzymes, Escherichia coli PBP5, Neisseria gonorrhoeae PBP4, and Streptococcus pneumoniae PBP3, and the LMMC enzymes, the Actinomadura R39 DD-peptidase, Bacillus subtilis PBP4a, and N. gonorrhoeae PBP3). The R39 enzyme (LMMC), like the previously studied Streptomyces R61 DD-peptidase (LMMB), specifically and rapidly hydrolyzes stem peptide fragments with a free N-terminus. In accord with this result, the crystal structures of the R61 and R39 enzymes display a binding site specific to the stem peptide N-terminus. These are water-soluble enzymes, however, with no known specific function in vivo. On the other hand, soluble versions of the remaining enzymes of those noted above, all of which are likely to be membrane-bound and/or associated in vivo and have been assigned particular roles in cell wall biosynthesis and maintenance, show little or no specificity for peptides containing elements of peptidoglycan structure. Peptidoglycan-mimetic boronate transition-state analogues do inhibit these enzymes but display notable specificity only for the LMMC enzymes, where, unlike peptide substrates, they may be able to effectively induce a specific active site structure. The manner in which LMMA (and HMM) DD-peptidases achieve substrate specificity, both in vitro and in vivo, remains unknown. [less ▲]

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See detailThe attachment of Bacillus amyloliquefaciens FZB42 to tomato roots is impaired in a mutant devoid of GalM and PBP4a.
Van der Heiden, Edwige; Jourdan, Emmanuel ULg; Delmarcelle, Michaël ULg et al

Poster (2011, June 20)

In vivo experiments on tomato (Lycopersicon esculentum) roots have been performed: drench of 10-day hydroponic cultures with bacterial suspensions. A second identical treatment was applied one week later ... [more ▼]

In vivo experiments on tomato (Lycopersicon esculentum) roots have been performed: drench of 10-day hydroponic cultures with bacterial suspensions. A second identical treatment was applied one week later and after 30 days of culture, the plants were collected and the bacteria detached from a precise weight of roots were diluted, plated and counted. The mutant devoid of both GalM and PBP4a (encoded by the galM and dacC operon) is affected in its capacity to colonize the tomato roots. [less ▲]

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See detailStructure-based design of benzoxazoles as new inhibitors for D-alanyl-D-alanine ligase
Tytgat, Isabelle; Vandevuer, Stéphane; Ortmans, Isabelle et al

in QSAR & Combinatorial Science (2009), 28(11-12), 1394-1404

d-Alanyl – d-alanine ligase is an enzyme which catalyzes the dimerization of d-alanine, and, as such, has an essential role in bacterial cell wall biosynthesis. It has been shown that inhibition of d ... [more ▼]

d-Alanyl – d-alanine ligase is an enzyme which catalyzes the dimerization of d-alanine, and, as such, has an essential role in bacterial cell wall biosynthesis. It has been shown that inhibition of d-alanyl – d-alanine ligase prevents bacterial growth. d-Alanyl – d-alanine ligase represents therefore a viable antimicrobial target. The 3D structure of this enzyme complexed with a phosphinophosphate inhibitor has been reported, which allows for structure-based design studies. Four softwares (LUDI, MCSS, Autodock, and Glide) developed either for fragment or full-molecule docking were compared and scored for their ability to position in the active site four prototypic ligands: two inhibitors, i.e. a phosphinophosphate derivative and d-cycloserine, d-alanine and d-alanyl – d-alanine. Best performances were obtained with Glide and MCSS. A short series of novel derivatives based on a 2-phenylbenzoxazole scaffold was designed de novo on the basis of computational data. The best compound was found to fully inhibit the d-alanyl – d-alanine ligase of E. faecalis with an IC50 of 400 mM. [less ▲]

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See detailProduction of four amyloidogenic variants of human lysozyme as inclusion bodies in Escherichia coli
Dumont, Janice ULg; Menzer, Linda ULg; Scarafone, Natacha ULg et al

Poster (2009)

Six variants of human lysozyme (I56T, F57I, W64R, D67H, F57I/T70N and W112R/T70N) are associated with a hereditary non-neuropathic systemic amyloidosis. This disease involved an extra cellular deposition ... [more ▼]

Six variants of human lysozyme (I56T, F57I, W64R, D67H, F57I/T70N and W112R/T70N) are associated with a hereditary non-neuropathic systemic amyloidosis. This disease involved an extra cellular deposition of amyloid fibrils made of lysozyme variants in a wide range of organs such as liver, spleen and kidneys [1]. The characterisation at the molecular level of two variants, I56T and D67H, has shown that these mutations reduce the stability and more particularly the global cooperativity of the protein. Consequently, under physiologically relevant conditions, these variants can transiently populate a partially unfolded state in which the beta-domain and the C-helix are cooperatively unfolded while the rest of the protein remains native like [1]. The formation of intermolecular interactions between the regions that are unfolded in this intermediate state is likely to be a fundamental trigger of the aggregation process that ultimately leads to the formation and deposition of fibrils in tissues. In order to study the effects of the other amyloidogenic mutations on the properties of lysozyme and thus to get more insight in the mechanism of amyloid formation, it is necessary to produce them in large quantities. The D67H, I56T and F57I variants are currently produced in Aspergillus niger; the expression in this organism is, however, time consuming and the yield is very low. The attempts to use alternative systems such as Pichia pastoris [2], Saccharomyces cerevisiae, and Arabidopsis thaliana have not been conclusive so far. In this work, we have produced the four single-point lysozyme variants as inclusion bodies in Escherichia coli and explored the possibility to refold them. [1] Dumoulin & al., (2006) Acc. Chem. Res., 39, 603 - 610 [2] Kumita & al., (2006) FEBS J., 273, 711-720 [less ▲]

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See detailCharacterization of the proteins encoded by the Bacillus subtilis yoxA-dacC operon.
Duez, Colette ULg; Zervosen, Astrid ULg; Teller, Nathalie et al

in FEMS Microbiology Letters (2009), 300

Abstract In Bacillus subtilis, the yoxA and dacC genes were proposed to form an operon. The yoxA gene was overexpressed in Escherichia coli and its product fused to a polyhistidine tag was purified. An ... [more ▼]

Abstract In Bacillus subtilis, the yoxA and dacC genes were proposed to form an operon. The yoxA gene was overexpressed in Escherichia coli and its product fused to a polyhistidine tag was purified. An aldose-1-epimerase or mutarotase activity was measured with the YoxA protein that we propose to rename as GalM by analogy with its counterpart in E. coli. The peptide d-Glu-delta-m-A(2)pm-d-Ala-m-A(2)pm-d-Ala mimicking the B. subtilis and E. coli interpeptide bridge was synthesized and incubated with the purified dacC product, the PBP4a. A clear dd-endopeptidase activity was obtained with this penicillin-binding protein, or PBP. The possible role of this class of PBP, present in almost all bacteria, is discussed. [less ▲]

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See detailThe linker region plays a key role in the adaptation to cold of the cellulase from an Antarctic bacterium
Sonan, Guillaume K; Receveur-Brechot, Véronique; Duez, Colette ULg et al

in Biochemical Journal (2007), 407(Part 2), 293-302

The psychrophilic cellulase, Cel5G, from the Antarctic bacterium Pseudoalteromonas haloplanktis is composed of a catalytic module (CM) joined to a carbohydrate-binding module (CBM) by an unusually long ... [more ▼]

The psychrophilic cellulase, Cel5G, from the Antarctic bacterium Pseudoalteromonas haloplanktis is composed of a catalytic module (CM) joined to a carbohydrate-binding module (CBM) by an unusually long, extended and flexible linker region (LR) containing three loops closed by three disulfide bridges. To evaluate the possible role of this region in cold adaptation, the LR was sequentially shortened by protein engineering, successively deleting one and two loops of this module, whereas the last disulfide bridge was also suppressed by replacing the last two cysteine residue by two alanine residues. The kinetic and thermodynamic properties of the mutants were compared with those of the full-length enzyme, and also with those of the cold-adapted CM alone and with those of the homologous mesophilic enzyme, Cel5A, from Erwinia chrysanthemi. The thermostability of the mutated enzymes as well as their relative flexibility were evaluated by differential scanning calorimetry and fluorescence quenching respectively. The topology of the structure of the shortest mutant was determined by SAXS (small-angle X-ray scattering). The data indicate that the sequential shortening of the LR induces a regular decrease of the specific activity towards macromolecular substrates, reduces the relative flexibility and concomitantly increases the thermostability of the shortened enzymes. This demonstrates that the long LR of the full-length enzyme favours the catalytic efficiency at low and moderate temperatures by rendering the structure not only less compact, but also less stable, and plays a crucial role in the adaptation to cold of this cellulolytic enzyme. [less ▲]

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See detailCrystal structure of the Bacillus subtilis penicillin-binding protein 4a, and its complex with a peptidoglycan mimetic peptide
Sauvage, Eric ULg; Duez, Colette ULg; Herman, Raphaël ULg et al

in Journal of Molecular Biology (2007), 371(2), 528-539

The genome of Bacillus subtilis encodes 16 penicillin-binding proteins (PBPs) involved in the synthesis and/or remodelling of the peptidoglycan during the complex life cycle of this sporulating Gram ... [more ▼]

The genome of Bacillus subtilis encodes 16 penicillin-binding proteins (PBPs) involved in the synthesis and/or remodelling of the peptidoglycan during the complex life cycle of this sporulating Gram-positive rod-shaped bacterium. PBP4a (encoded by the dacC gene) is a low-molecular mass PBP clearly exhibiting in vitro DD-carboxypeptidase activity. We have solved the crystal structure of this protein alone and in complex with a peptide (D-alpha'-aminopymelyl-epsilon-D-alanyl-D-alanine) that mimics the C-terminal end of the Bacillus peptidoglycan stem peptide. PBP4a is composed of three domains: the penicillin-binding domain with a fold similar to the class A 13-lactamase structure and two domains inserted between the conserved motifs 1 and 2 characteristic of the penicillin-recognizing enzymes. The soaking of PBP4a in a solution Of D-alpha-aminopymelyl-epsilon-D-alanyl-D-alanine resulted in an adduct between PBP4a and a D-alpha-aminopimelyl-epsilon-D-alanine dipeptide and an unbound D-alanine, i.e. the products of acylation of PBP4a by D-alpha-aminopymelyl-epsilon-D-alanyl-D-alanine with the release of a D-alanine. The adduct also reveals a binding pocket specific to the diaminopimelic acid, the third residue of the peptidoglycan stem pentapeptide of B. subtilis. This pocket is specific for this class of PBPs. (C) 2007 Elsevier Ltd. All rights reserved. [less ▲]

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See detailSolubilisation and binding characteristics of a recombinant beta(2)-adrenergic receptor expressed in the membrane of Escherichia coli for the multianalyte detection of beta-agonists and antagonists residues in food-producing animals
Danyi, Sophie ULg; Degand, Guy ULg; Duez, Colette ULg et al

in Analytica Chimica Acta (2007), 589(2), 159-165

The number of substances with beta-agonistic activity, illegally introduced in meat production or in sports doping as anabolic or beta-blocking agents is increasing. Analytical methods suited for their ... [more ▼]

The number of substances with beta-agonistic activity, illegally introduced in meat production or in sports doping as anabolic or beta-blocking agents is increasing. Analytical methods suited for their multianalyte detection are thus necessary. In this perspective, receptor assays were developed. The research activities undertaken in this study describe the solubilisation of a recombinant human beta(2)-adrenergic receptor produced in the inner membrane of genetically modified Escherichia coli, using the detergent n-dodecyl-beta-D-maltoside. Its potential to detect the presence of beta-agonists or beta-blockers in biological samples was evaluated. The solubilised beta(2)-adrenergic receptor retained its binding affinity in a radio-receptor assay based on the competition for the binding to receptors between a ligand (beta-agonist or antagonist) and the radioligand [I-125]iodocyanopindolol. The IC50 values ranged from 5 +/- x 10(-8) M (clenbuterol) to 8 +/- 2 x 10(-6) M (isoxsuprine) for the beta-agonists tested and from 1.5 +/- 0.2 x 10(-10) M (carazolol) to 1.2 +/- 0.2 x 10(-5) M (metoprolol) for the beta-blockers tested. It was shown to have a lower limit of detection than a radio-receptor assay using the solubilised beta(2)-adrenoceptor expressed in a mammalian cell line. The solubilised recombinant human beta(2)-adrenoreceptor expressed in E. coli would be a useful tool to develop non radioactive multianalyte screening methods. (c) 2007 Elsevier B.V. All rights reserved. [less ▲]

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See detailMolecular characterisation of a versatile peroxidase from a Bjerkandera strain
Moreira, Patricia R.; Duez, Colette ULg; Dehareng, Dominique ULg et al

in Journal of Biotechnology (2005), 118(4), 339-352

The cloning and sequencing of the rbpa gene coding for a versatile peroxidase from a novel Bjerkandera strain is hereby reported. The 1777 bp isolated fragment contained a 1698 bp peroxidase-encoding gene ... [more ▼]

The cloning and sequencing of the rbpa gene coding for a versatile peroxidase from a novel Bjerkandera strain is hereby reported. The 1777 bp isolated fragment contained a 1698 bp peroxidase-encoding gene, interrupted by 11 introns. The 367 amino acid-deduced sequence includes a 27 amino acid-signal peptide. The molecular model, built via homology modelling with crystal structures of four fungal peroxidases, highlighted the amino acid residues putatively involved in manganese binding and aromatic substrate oxidation. The potential heme pocket residues (R44, F47, H48, E79, N85, H177, F194 and D239) include both distal and proximal histidines (H48 and H177). RBP possesses potential calcium-binding residues (D49, G67, D69, S71, S178, D195, T197, I200 and D202) and eight cysteine residues (C3, C15, C16, C35, C121, C250, C286, C316). In addition, RIBP includes residues involved in substrate oxidation: three acidic residues (E37, E41 and D183)-putatively involved in manganese binding and H83 and W172-potentially involved in oxidation of aromatic substrates. Characterisation of nucleotide and amino acid sequences include RBP in versatile peroxidase group sharing catalytic properties of both UP and MnP. In addition, the RBP enzyme appears to be closely related with the ligninolytic peroxidases from the Trametes versicolor strain. (C) 2005 Published by Elsevier B.V. [less ▲]

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See detailCrystal structure of the Actinomadura R39 DD-peptidase reveals new domains in penicillin-binding proteins.
Sauvage, Eric ULg; Herman, Raphaël ULg; Petrella, Stephanie et al

in Journal of Biological Chemistry (2005), 280(35), 31249-56

Actinomadura sp. R39 produces an exocellular DD-peptidase/penicillin-binding protein (PBP) whose primary structure is similar to that of Escherichia coli PBP4. It is characterized by a high beta-lactam ... [more ▼]

Actinomadura sp. R39 produces an exocellular DD-peptidase/penicillin-binding protein (PBP) whose primary structure is similar to that of Escherichia coli PBP4. It is characterized by a high beta-lactam-binding activity (second order rate constant for the acylation of the active site serine by benzylpenicillin: k2/K = 300 mm(-1) s(-1)). The crystal structure of the DD-peptidase from Actinomadura R39 was solved at a resolution of 1.8 angstroms by single anomalous dispersion at the cobalt resonance wavelength. The structure is composed of three domains: a penicillin-binding domain similar to the penicillin-binding domain of E. coli PBP5 and two domains of unknown function. In most multimodular PBPs, additional domains are generally located at the C or N termini of the penicillin-binding domain. In R39, the other two domains are inserted in the penicillin-binding domain, between the SXXK and SXN motifs, in a manner similar to "Matryoshka dolls." One of these domains is composed of a five-stranded beta-sheet with two helices on one side, and the other domain is a double three-stranded beta-sheet inserted in the previous domain. Additionally, the 2.4-angstroms structure of the acyl-enzyme complex of R39 with nitrocefin reveals the absence of active site conformational change upon binding the beta-lactams. [less ▲]

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See detailThe ponA gene of Enterococcus faecalis JH2-2 codes for a low-affinity class a penicillin-binding protein
Duez, Colette ULg; Hallut, Séverine; Rhazi, Noureddine ULg et al

in Journal of Bacteriology (2004), 186(13), 4412-4416

soluble derivative of the Enterococcus faecalis JH2-2 class A PBP1 (*PBP1) was overproduced and purified. It exhibited a glycosyltransferase activity on the Escherichia coli (14)C(-)labeled lipid 11 ... [more ▼]

soluble derivative of the Enterococcus faecalis JH2-2 class A PBP1 (*PBP1) was overproduced and purified. It exhibited a glycosyltransferase activity on the Escherichia coli (14)C(-)labeled lipid 11 precursor. As a DD-peptidase, it could hydrolyze thiolester substrates with efficiencies similar to those of other class A penicillin-binding proteins (PBPs) and bind beta-lactams, but with k(2)/K (a parameter accounting for the acylation step efficiency) values characteristic of penicillin-resistant PBPs. [less ▲]

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See detailThe Penicillin Resistance of Enterococcus Faecalis Jh2-2r Results from an Overproduction of the Low-Affinity Penicillin-Binding Protein Pbp4 and Does Not Involve a Psr-Like Gene
Duez, Colette ULg; Zorzi, Willy ULg; Sapunaric, Frédéric ULg et al

in Microbiology (2001), 147(Pt 9), 2561-9

A penicillin-resistant mutant, JH2-2r (MIC 75 microg ml(-1)), was isolated from Enterococcus faecalis JH2-2 (MIC 5 microg ml(-1)) by successive passages on plates containing increasing concentrations of ... [more ▼]

A penicillin-resistant mutant, JH2-2r (MIC 75 microg ml(-1)), was isolated from Enterococcus faecalis JH2-2 (MIC 5 microg ml(-1)) by successive passages on plates containing increasing concentrations of benzylpenicillin. A comparison of the penicillin-binding protein (PBP) profiles in the two strains revealed a more intensely labelled PBP4 in JH2-2r. Because the sequences of the JH2-2 and JH2-2r pbp4 genes were strictly identical, even in their promoter regions, this intensive labelling could only be associated with an overproduction of the low-affinity PBP4. No psr gene analogous to that proposed to act as a regulator of PBP5 synthesis in Enterococcus hirae and Enterococcus faecium could be identified in the vicinity of pbp4 in E. faecalis JH2-2 and JH2-2r. However, a psr-like gene distant from pbp4 was identified. The cloning and sequencing of that psr-like gene from both E. faecalis strains indicated that they were identical. It is therefore postulated that the PBP4 overproduction in E. faecalis JH2-2r results from the modification of an as yet unidentified factor. [less ▲]

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

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

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

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

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See 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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