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See detailHow to build a biological linker dedicated to the engineering of novel drug delivery systems
Crasson, Oscar ULg; Galleni, Moreno ULg; Parente, Raffaella et al

Poster (2014, May 15)

Nowadays, chemical linkages are widely used in industry to coat bioactive molecules on biocompatible supports. However, some issues inherent to the linkage procedure remain. For example, chemical ... [more ▼]

Nowadays, chemical linkages are widely used in industry to coat bioactive molecules on biocompatible supports. However, some issues inherent to the linkage procedure remain. For example, chemical reactions often damage the structure and therefore the activity of the immobilized molecules. In this work, we propose the use of a human chitin binding domain (ChBD) to immobilized bioactive molecules on a polysaccharide-based surface. ChBD belongs to the human chitinase called chitotriosidase and was shown to interact specifically with chitin and chitooligomers. Using molecular biology and protein engineering, we have developped of a new biological tool based on the hybrid protein technology. In this technology, we can create chimeric proteins that have the ability to bind polysaccharidic supports thanks to their binding domain that can be covalently linked with any bioactive molecule that can confer prophylactic or therapeutic activities. In this study, we have successfully shown that we can use the chitin binding domain (ChBD) from the human chitotriosidase as a biological linker dedicated to the engineering of novel drug delivery systems containing chitin or other analogous polysaccharides. [less ▲]

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See detailCrystal Structure of the Extended-Spectrum β -Lactamase PER-2 and Insights into the Role of Specific Residues in the Interaction with β -Lactams and β -Lactamase Inhibitors
Ruggiero, Melina; Kerff, Frédéric ULg; Herman, Raphaël ULg et al

in Antimicrobial Agents and Chemotherapy (2014), 58(10), 5994-6002

PER-2 belongs to a small (7 members to date) group of extended-spectrum beta-lactamases. It has 88% amino acid identity with PER-1 and both display high catalytic efficiencies toward most beta-lactams. In ... [more ▼]

PER-2 belongs to a small (7 members to date) group of extended-spectrum beta-lactamases. It has 88% amino acid identity with PER-1 and both display high catalytic efficiencies toward most beta-lactams. In this study, we determined the X-ray structure of PER-2 at 2.20 A and evaluated the possible role of several residues in the structure and activity toward beta-lactams and mechanism-based inhibitors. PER-2 is defined by the presence of a singular trans bond between residues 166 to 167, which generates an inverted Omega loop, an expanded fold of this domain that results in a wide active site cavity that allows for efficient hydrolysis of antibiotics like the oxyimino-cephalosporins, and a series of exclusive interactions between residues not frequently involved in the stabilization of the active site in other class A beta-lactamases. PER beta-lactamases might be included within a cluster of evolutionarily related enzymes harboring the conserved residues Asp136 and Asn179. Other signature residues that define these enzymes seem to be Gln69, Arg220, Thr237, and probably Arg/Lys240A ("A" indicates an insertion according to Ambler's scheme for residue numbering in PER beta-lactamases), with structurally important roles in the stabilization of the active site and proper orientation of catalytic water molecules, among others. We propose, supported by simulated models of PER-2 in combination with different beta-lactams, the presence of a hydrogen-bond network connecting Ser70-Gln69-water-Thr237-Arg220 that might be important for the proper activity and inhibition of the enzyme. Therefore, we expect that mutations occurring in these positions will have impacts on the overall hydrolytic behavior. [less ▲]

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See detailGenetic and kinetic characterization of the novel AmpC beta-lactamases DHA-6 and DHA-7.
Perez-Llarena, Francisco Jose; Zamorano, Laura; Kerff, Frédéric ULg et al

in Antimicrobial agents and chemotherapy (2014)

During a Spanish surveillance study, two natural variants of DHA beta-lactamases, DHA-6 and DHA-7 were found, with the replacements Ala226Thr and Phe322Ser respect to DHA-1, respectively. The enzymes were ... [more ▼]

During a Spanish surveillance study, two natural variants of DHA beta-lactamases, DHA-6 and DHA-7 were found, with the replacements Ala226Thr and Phe322Ser respect to DHA-1, respectively. The enzymes were isolated from Escherichia coli and Enterobacter cloacae clinical isolates, respectively. The aim of the study was the genetic, microbiological and biochemical characterization of the DHA-6 and DHA-7 beta-lactamases. The blaDHA-6 andblaDHA-7 genes were located in I1 and HI2 incompatibility group plasmids of 87.3 and 310.4 kb, respectively. The gene context of both blaDHA-6 andblaDHA-7 was similar to that already described for blaDHA-1 gene and included the qnrB4 and aadA genes. The MICs for cephalothin, aztreonam, cefotaxime and ceftazidime were 8 to 30 fold lower for the DHA-6 than for DHA-1 and DHA-7 expressed in the same isogenic E.coli TG1 strain. Interestingly the MIC for cefoxitin was higher in DHA-6 expressing transformant compared to DHA-1 and DHA-7. Biochemical studies with pure beta-lactamases revealed a slightly lower catalytic efficiency of DHA-6 against cephalothin, ceftazidime and cefotaxime compared to DHA-1 and DHA-7. To understand this behavior, stability experiments were carried out and showed that the DHA-6 protein displayed a significantly higher stability than DHA-1 and DHA-7 enzymes. The proximity of Thr226 to the N-terminal in the tertiary protein structure in DHA-6 may promote this stabilization and consequently could induce a slight reduction of the dynamic of this enzyme primarily affecting the hydrolysis of some of the bulkiest antibiotics. [less ▲]

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See detailNew mutations in ADC-type beta-lactamases from Acinetobacter spp. affect cefoxitin and ceftazidime hydrolysis.
Perez, Astrid; Perez-Llarena, Francisco Jose; Garcia, Patricia et al

in The Journal of antimicrobial chemotherapy (2014)

OBJECTIVES: Two natural variants of ADC-type beta-lactamases of Acinetobacter spp., ADC-1 and ADC-5, differ by nine mutations in their protein sequence. ADC-5 hydrolyses cefoxitin better than ADC-1 and ... [more ▼]

OBJECTIVES: Two natural variants of ADC-type beta-lactamases of Acinetobacter spp., ADC-1 and ADC-5, differ by nine mutations in their protein sequence. ADC-5 hydrolyses cefoxitin better than ADC-1 and the opposite is true for ceftazidime. We produced single and combined mutations in ADC-5 and characterized the variants microbiologically and biochemically to determine which amino acid residues are involved in the hydrolysis of beta-lactam antibiotics in this family of beta-lactamases. METHODS: Site-directed mutagenesis, with blaADC-5 as a source of DNA, was used to generate nine single mutated and three combined mutated enzymes. The proteins (wild-type and derivatives) were then expressed in isogenic conditions in Escherichia coli. MICs of beta-lactams were determined using Etest strips. ADC-1, ADC-5, ADC-5-P167S and ADC-5-P167S/D242G/Q163K/G342R were also purified and the kinetic parameters determined for ceftazidime, cefoxitin, cefalotin and ampicillin. RESULTS: Single mutations did not significantly convert the hydrolysis spectrum of the ADC-5 enzyme into that of the ADC-1 enzyme, although among all studied mutants only the quadruple mutant (ADC-5-P167S/D242G/Q163K/G342R) displayed microbiological and biochemical properties consistent with those of ADC-1. CONCLUSIONS: Although some single mutations are known to affect cefepime hydrolysis in ADC-type beta-lactamases, little is known about ceftazidime and cefoxitin hydrolysis in this family of beta-lactamases. Hydrolysis of these antibiotics appears to be positively and negatively affected, respectively, by the Q163K, P167S, D242G and G342R amino acid replacements. [less ▲]

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See detailDer p 1 is the primary activator of Der p 3, Der p 6 and Der p 9 the proteolytic allergens produced by the house dust mite Dermatophagoides pteronyssinus
Herman, Julie ULg; Thelen, Nicolas ULg; Smargiasso, Nicolas ULg et al

in Biochimica et Biophysica Acta - General Subjects (2013), 1840

Background: The enzymatic activity of the four proteases found in the house dust mite Dermatophagoides pteronyssinus is involved in the pathogenesis of allergy. Our aim was to elucidate the activation ... [more ▼]

Background: The enzymatic activity of the four proteases found in the house dust mite Dermatophagoides pteronyssinus is involved in the pathogenesis of allergy. Our aim was to elucidate the activation cascade of their corresponding precursor forms and particularly to highlight the interconnection between proteases during this cascade. Methods: The cleavage of the four peptides corresponding to the mite zymogen activation sites was studied on the basis of the Förster Resonance Energy Transfermethod. The proDer p 6 zymogen was then produced in Pichia pastoris to elucidate its activation mechanismbymite proteases, especially Der p 1. The role of the propeptide in the inhibition of the enzymatic activity of Der p 6 was also examined. Finally, the Der p 1 and Der p 6 proteases were localised via immunolocalisation in D. pteronyssinus. Results: All peptides were specifically cleaved by Der p 1, such as proDer p 6. The propeptide of proDer p 6 inhibited the proteolytic activity of Der p 6, but once cleaved, it was degraded by the protease. The Der p 1 and Der p 6 proteases were both localised to the midgut of the mite. Conclusions: Der p 1 in either its recombinant formor in the natural context of house dustmite extracts specifically cleaves all zymogens, thus establishing its role as a major activator of both mite cysteine and serine proteases. General significance: This finding suggests that Der p 1 may be valuable target against mites. [less ▲]

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See detailClass A β -Lactamases as Versatile Scaffolds to Create Hybrid Enzymes: Applications from Basic Research to Medicine
Huynen, Céline ULg; Filée, Patrice; Matagne, André ULg et al

in BioMed Research International (2013), 2013

Designing hybrid proteins is a major aspect of protein engineering and covers a very wide range of applications frombasic research to medical applications. This review focuses on the use of class A 𝛽 ... [more ▼]

Designing hybrid proteins is a major aspect of protein engineering and covers a very wide range of applications frombasic research to medical applications. This review focuses on the use of class A 𝛽-lactamases as versatile scaffolds to design hybrid enzymes (referred to as 𝛽-lactamase hybrid proteins, BHPs) in which an exogenous peptide, protein or fragment thereof is inserted at various permissive positions.We discuss how BHPs can be specifically designed to create bifunctional proteins, to produce and to characterize proteins that are otherwise difficult to express, to determine the epitope of specific antibodies, to generate antibodies against nonimmunogenic epitopes, and to better understand the structure/function relationship of proteins. [less ▲]

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See detailA Pathway closely related to the D-tagatose pathway of Gram-Negative Enterobacteria Identified in the Gram-Positive Bacterium Bacillus licheniformis
Van Der Heiden, Edwige ULg; Delmarcelle, Michaël ULg; Lebrun, Sarah ULg et al

Poster (2013, June)

We report the first identification of a gene cluster involved in d-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the d-tagatose pathway of the Gram-negative bacterium ... [more ▼]

We report the first identification of a gene cluster involved in d-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the d-tagatose pathway of the Gram-negative bacterium Klebsiella oxytoca, in contrast to the d-tagatose 6-phosphate pathway described in the Gram-positive bacterium Staphylococcus aureus. [less ▲]

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See detailA Pathway closely related to the D-tagatose pathway of Gram-Negative Enterobacteria Identified in the Gram-Positive Bacterium Bacillus licheniformis
Van Der Heiden, Edwige ULg; Delmarcelle, Michaël ULg; Lebrun, Sarah ULg et al

in Applied and Environmental microbiology (2013), 79(11), 3511-3515

We report the first identification of a gene cluster involved in d-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the d-tagatose pathway of the Gram-negative bacterium ... [more ▼]

We report the first identification of a gene cluster involved in d-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the d-tagatose pathway of the Gram-negative bacterium Klebsiella oxytoca, in contrast to the d-tagatose 6-phosphate pathway described in the Gram-positive bacterium Staphylococcus aureus. [less ▲]

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See detailAllosteric inhibition of VIM metallo-beta-lactamase by a camelid nanobody
Sohier, Jean ULg; Laurent, Clémentine ULg; Chevigné, Andy et al

in Biochemical Journal (2013), 450(3), 477-486

Metallo-β-lactamase (MβL) enzymes are usually produced by multiresistant Gram-negative bacterial strains and have spread worldwide. An approach based on phage display was employed to select single-domain ... [more ▼]

Metallo-β-lactamase (MβL) enzymes are usually produced by multiresistant Gram-negative bacterial strains and have spread worldwide. An approach based on phage display was employed to select single-domain antibody fragments (VHHs also called Nanobodies) that would inhibit the clinically relevant VIM-4 MβL. Out of more than 50 selected nanobodies, only the NbVIM_38 nanobody inhibited VIM-4. The paratope, inhibition mechanism and epitope of NbVIM_38 nanobody were then characterised. An alanine scan of the NbVIM_38 paratope showed that its binding was driven by hydrophobic amino acids. The inhibitory concentration was in the µM range for all tested β-lactams. In addition, the inhibition was found to follow a mixed hyperbolic profile with a predominantly uncompetitive component. Moreover, substrate inhibition was recorded only after nanobody binding. These kinetic data are indicative of a binding site that is distant from the active site. This finding was confirmed by epitope mapping analysis that was performed using peptides, and which identified two stretches of amino acids in the L6 loop and at the end of the alpha2 helix. Because this binding site is distant from the active site and alters both the substrate binding and catalytic properties of VIM-4, this nanobody can be considered as an allosteric inhibitor. [less ▲]

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See detailBiochemical and Structural studies of the type I tagatose bisphosphate aldolases
Freichels, Régine ULg; Guarino, Carla; Delmarcelle, Michaël ULg et al

Poster (2013, February 26)

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See detailThe proline-rich motif of the proDer p 3 allergen propeptide is crucial for protease-protease interaction.
Dumez, Marie-Eve ULg; Herman, Julie; Campisi, Vincenzo ULg et al

in PloS one (2013), 8(9), 68014

The majority of proteases are synthesized in an inactive form, termed zymogen, which consists of a propeptide and a protease domain. The propeptide is commonly involved in the correct folding and specific ... [more ▼]

The majority of proteases are synthesized in an inactive form, termed zymogen, which consists of a propeptide and a protease domain. The propeptide is commonly involved in the correct folding and specific inhibition of the enzyme. The propeptide of the house dust mite allergen Der p 3, NPILPASPNAT, contains a proline-rich motif (PRM), which is unusual for a trypsin-like protease. By truncating the propeptide or replacing one or all of the prolines in the non-glycosylated zymogen with alanine(s), we demonstrated that the full-length propeptide is not required for correct folding and thermal stability and that the PRM is important for the resistance of proDer p 3 to undesired proteolysis when the protein is expressed in Pichia pastoris. Additionally, we followed the maturation time course of proDer p 3 by coupling a quenched-flow assay to mass spectrometry analysis. This approach allowed to monitor the evolution of the different species and to determine the steady-state kinetic parameters for activation of the zymogen by the major allergen Der p 1. This experiment demonstrated that prolines 5 and 8 are crucial for proDer p 3-Der p 1 interaction and for activation of the zymogen. [less ▲]

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See detailKinetic and crystallographic studies of extended-spectrum GES-11, GES-12, and GES-14 β-lactamases.
Delbrück, Heinrich; Bogaerts, Pierre; Kupper, Michaël et al

in Antimicrobial Agents and Chemotherapy (2012), 56(11)

GES-1 is a class A extended-spectrum β-lactamase conferring resistance to penicillins, narrow- and expanded-spectrum cephalosporins, and ceftazidime. However, GES-1 poorly hydrolyzes aztreonam and ... [more ▼]

GES-1 is a class A extended-spectrum β-lactamase conferring resistance to penicillins, narrow- and expanded-spectrum cephalosporins, and ceftazidime. However, GES-1 poorly hydrolyzes aztreonam and cephamycins and exhibits very low k(cat) values for carbapenems. Twenty-two GES variants have been discovered thus far, differing from each other by 1 to 3 amino acid substitutions that affect substrate specificity. GES-11 possesses a Gly243Ala substitution which seems to confer to this variant an increased activity against aztreonam and ceftazidime. GES-12 differs from GES-11 by a single Thr237Ala substitution, while GES-14 differs from GES-11 by the Gly170Ser mutation, which is known to confer increased carbapenemase activity. GES-11 and GES-12 were kinetically characterized and compared to GES-1 and GES-14. Purified GES-11 and GES-12 showed strong activities against most tested β-lactams, with the exception of temocillin, cefoxitin, and carbapenems. Both variants showed a significantly increased rate of hydrolysis of cefotaxime, ceftazidime, and aztreonam. On the other hand, GES-11 and GES-12 (and GES-14) variants all containing Ala243 exhibited increased susceptibility to classical inhibitors. The crystallographic structures of the GES-11 and GES-14 β-lactamases were solved. The overall structures of GES-11 and GES-14 are similar to that of GES-1. The Gly243Ala substitution caused only subtle local rearrangements, notably in the typical carbapenemase disulfide bond. The active sites of GES-14 and GES-11 are very similar, with the Gly170Ser substitution leading only to the formation of additional hydrogen bonds of the Ser residue with hydrolytic water and the Glu166 residue. [less ▲]

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