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See detailStudy of a Natural Mutant SHV-Type beta-Lactamase, SHV-104, from Klebsiella pneumoniae
Achour, Nahed Ben; Belhadj, Omrane; Galleni, Moreno ULg et al

in International journal of microbiology (2014), 2014

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See detailDevelopment of recombinant stable house dust mite allergen Der p 3 molecules for component-resolved diagnosis and specific immunotherapy.
Bouaziz, Ahlem; Walgraffe, David; Bouillot, Celine et al

in Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology (2014)

BACKGROUND: The allergen Der p 3 is underrepresented in house dust mite (HDM) extracts probably due to autolysis. Recombinant stable molecule of the allergen is thus needed to improve the diagnosis of ... [more ▼]

BACKGROUND: The allergen Der p 3 is underrepresented in house dust mite (HDM) extracts probably due to autolysis. Recombinant stable molecule of the allergen is thus needed to improve the diagnosis of allergy and the safety and efficacy of immunotherapy. OBJECTIVE: The current study reports the immunological characterization of two recombinant molecules of the HDM allergen Der p 3 as useful tools for diagnosis and immunotherapy. METHODS: Recombinant mature (rDer p 3) and immature (proDer p 3) Der p 3 and their corresponding S196A mutants were produced in Pichia pastoris and purified. The stability, IgE-binding capacity and allergenicity of the different proteins were analyzed and compared with those of the major mite allergen Der p 1 used as a reference. Additionally, the immunogenicity of the different allergens was evaluated in a murine model of Der p 3 sensitization. RESULTS: Compared to the IgE reactivity to recombinant and natural Der p 3 (nDer p 3), the mean IgE binding of patient's sera to rDer p 3-S196A (50 %) was higher. The poorly binding to nDer p 3 or rDer p 3 was due to autolysis of the allergen. Contrary to Der p 3, proDer p 3 displayed very weak IgE reactivity, as measured by sandwich ELISA and competitive inhibition, RBL degranulation and human basophil activation assays. Moreover, proDer p 3 induced a TH 1-biased immune response that prevented an allergic response in mice but retained Der p 3-specific T cell reactivity. CONCLUSION: rDer p 3-S196A should be used for the diagnosis of HDM allergy elicited by Der p 3, and proDer p 3 may represent a hypoallergen of Der p 3. This article is protected by copyright. All rights reserved. [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 detailNovel Cold-Adapted Esterase MHlip from an Antarctic Soil Metagenome.
Berlemont, Renaud; Jacquin, Olivier; Delsaute, Maud et al

in Biology (2013), 2(1), 177-88

An Antarctic soil metagenomic library was screened for lipolytic enzymes and allowed for the isolation of a new cytosolic esterase from the a/b hydrolase family 6, named MHlip. This enzyme is related to ... [more ▼]

An Antarctic soil metagenomic library was screened for lipolytic enzymes and allowed for the isolation of a new cytosolic esterase from the a/b hydrolase family 6, named MHlip. This enzyme is related to hypothetical genes coding esterases, aryl-esterases and peroxydases, among others. MHlip was produced, purified and its activity was determined. The substrate profile of MHlip reveals a high specificity for short p-nitrophenyl-esters. The apparent optimal activity of MHlip was measured for p-nitrophenyl-acetate, at 33 degrees C, in the pH range of 6-9. The MHlip thermal unfolding was investigated by spectrophotometric methods, highlighting a transition (Tm) at 50 degrees C. The biochemical characterization of this enzyme showed its adaptation to cold temperatures, even when it did not present evident signatures associated with cold-adapted proteins. Thus, MHlip adaptation to cold probably results from many discrete structural modifications, allowing the protein to remain active at low temperatures. Functional metagenomics is a powerful approach to isolate new enzymes with tailored biophysical properties (e.g., cold adaptation). In addition, beside the ever growing amount of sequenced DNA, the functional characterization of new catalysts derived from environment is still required, especially for poorly characterized protein families like alpha/b hydrolases. [less ▲]

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See detailR164H and V240H replacements by site-directed mutagenesis of TEM-149 extended-spectrum beta-lactamase: kinetic analysis of TEM-149H240 and TEM-149H164-H240 laboratory mutants.
Perilli, Mariagrazia; Celenza, Giuseppe; Mercuri, Paola ULg et al

in Antimicrobial agents and chemotherapy (2013), 57(2), 1047-9

Two laboratory mutant forms, TEM-149(H240) and TEM-149(H164-H240), of the TEM-149 extended-spectrum beta-lactamase enzyme were constructed by site-directed mutagenesis. TEM-149(H240) and TEM-149(H164-H240 ... [more ▼]

Two laboratory mutant forms, TEM-149(H240) and TEM-149(H164-H240), of the TEM-149 extended-spectrum beta-lactamase enzyme were constructed by site-directed mutagenesis. TEM-149(H240) and TEM-149(H164-H240) were similar in kinetic behavior, except with respect to benzylpenicillin and ceftazidime. Molecular modeling of the two mutant enzymes demonstrated the role of histidine at position 240 in the reduction of the affinity of the enzyme for ceftazidime. [less ▲]

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See detailThree-dimensional structure of RBcel1, a metagenome-derived psychrotolerant family GH5 endoglucanase.
Delsaute, Maud; Berlemont, Renaud; Dehareng, Dominique et al

in Acta crystallographica. Section F, Structural biology and crystallization communications (2013), 69(Pt 8), 828-33

RBcel1 is an endoglucanase belonging to glycoside hydrolase family 5 subfamily 5 (GH5_5) that was recently identified from a soil metagenome library from the Antarctic. Unlike its closest structural ... [more ▼]

RBcel1 is an endoglucanase belonging to glycoside hydrolase family 5 subfamily 5 (GH5_5) that was recently identified from a soil metagenome library from the Antarctic. Unlike its closest structural homologue (Cel5A from Thermoascus aurantiacus), this enzyme was reported to be able to catalyze transglycosylation reactions and has putatively been implicated in the bacterial cellulose-synthesis process. Here, the structure of RBcel1 at 1.4 A resolution, solved by molecular replacement, is reported. The structure and putative substrate-binding site are described and compared with those of other GH5_5 subfamily members. [less ▲]

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See detailNovel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification
Berlemont, Renaud; Spee, Olivier; Delsaute, Maud et al

in Revista Argentina de Microbiologia (2013), (45), 3-12

in order to isolate novel organic solvent-tolerant (oSt) lipases, a metagenomic library was built using dna derived from a temperate forest soil sample. a two-step activity-based screening allowed the ... [more ▼]

in order to isolate novel organic solvent-tolerant (oSt) lipases, a metagenomic library was built using dna derived from a temperate forest soil sample. a two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid pRBest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. the deduced amino acid sequence (RBest1) contains the conserved lipolytic enzyme signature and is related to the previously described oSt lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 a/b hydrolase subgroup (abh04.04). Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenyl-esters, respectively, revealed that the enzyme is highly specific for butyrate (c4) compounds, behaving as an esterase rather than a lipase. the RBest1 esterase was purified and biochemically characterized. the optimal esterase activity was observed at ph 6.5 and at temperatures ranging from 38 to 45 °c. enzymatic activity, determined by hydrolysis of p‐nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. noteworthy, RBest1 remains significantly active at high ionic strength. these findings suggest that RBest1 possesses the ability of oSt enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins. [less ▲]

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