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See detailThe CphAII protein from Aquifex aeolicus exhibits a metal-dependent phosphodiesterase activity
Kupper, Michaël; Bauvois, Cédric; Frère, Jean-Marie ULg et al

in Extremophiles : Life Under Extreme Conditions (2012), 16(1)

The CphAII protein from the hyperthermophile Aquifex aeolicus shows the five conserved motifs of the metallo-β-lactamase (MBL) superfamily and presents 28% identity with the Aeromonas hydrophila subclass ... [more ▼]

The CphAII protein from the hyperthermophile Aquifex aeolicus shows the five conserved motifs of the metallo-β-lactamase (MBL) superfamily and presents 28% identity with the Aeromonas hydrophila subclass B2 CphA MBL. The gene encoding CphAII was amplified by PCR from the A. aeolicus genomic DNA and overexpressed in Escherichia coli using a pLex-based expression system. The recombinant CphAII protein was purified by a combination of heating (to denature E. coli proteins) and two steps of immobilized metal affinity chromatography. The purified enzyme preparation did not exhibit a β-lactamase activity but showed a metal-dependent phosphodiesterase activity versus bis-p-nitrophenyl phosphate and thymidine 5'-monophosphate p-nitrophenyl ester, with an optimum at 85°C. The circular dichroism spectrum was in agreement with the percentage of secondary structures characteristic of the MBL αββα fold. [less ▲]

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See detailNovel fragments of clavulanate observed in the structure of the class A b-lactamase from Bacillus licheniformis BS3
Power, Pablo; Mercuri, Paola ULg; Herman, Raphaël ULg et al

in Journal of Antimicrobial Chemotherapy (2012), 67(10), 2379-2387

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See detailCellulase involvement in the bacterial cellulose biosynthesis
Delsaute, Maud ULg; Berlemont, Renaud ULg; Bauvois, Cédric et al

Poster (2012)

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See detailOXA-198, an acquired carbapenem-hydrolyzing class D beta-lactamase from Pseudomonas aeruginosa.
El Garch, Farid; Bogaerts, Pierre; Bebrone, Carine ULg et al

in Antimicrobial Agents and Chemotherapy (2011), 55(10)

A carbapenem-resistant Pseudomonas aeruginosa strain (PA41437) susceptible to expanded-spectrum cephalosporins was recovered from several consecutive lower-respiratory-tract specimens of a patient who ... [more ▼]

A carbapenem-resistant Pseudomonas aeruginosa strain (PA41437) susceptible to expanded-spectrum cephalosporins was recovered from several consecutive lower-respiratory-tract specimens of a patient who developed a ventilator-associated pneumonia while hospitalized in an intensive care unit. Cloning experiments identified OXA-198, a new class D β-lactamase which was weakly related (less than 45% amino acid identity) to other class D β-lactamases. Expression in Escherichia coli TOP10 and in P. aeruginosa PAO1 led to transformants that were resistant to ticarcillin and showed reduced susceptibility to carbapenems and cefepime. The bla(OXA-198) gene was harbored by a class 1 integron carried by a ca. 46-kb nontypeable plasmid. This study describes a novel class D β-lactamase involved in carbapenem resistance in P. aeruginosa. [less ▲]

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See detailBroad antibiotic resistance profile of the subclass B3 metallo-β-lactamase GOB-1, a di-zinc enzyme.
Horsfall, Louise; Izougarhane, Youssef; Lassaux, Patricia et al

in FEBS Journal (2011), 278(8)

The metallo-β-lactamase (MBL) GOB-1 was expressed via a T7 expression system in Escherichia coli BL21(DE3). The MBL was purified to homogeneity and shown to exhibit a broad substrate profile, hydrolyzing ... [more ▼]

The metallo-β-lactamase (MBL) GOB-1 was expressed via a T7 expression system in Escherichia coli BL21(DE3). The MBL was purified to homogeneity and shown to exhibit a broad substrate profile, hydrolyzing all the tested β-lactam compounds efficiently. The GOB enzymes are unique among MBLs due to the presence of a glutamine residue at position 116, a zinc-binding residue in all known class B1 and B3 MBL structures. Here we produced and studied the Q116A, Q116N and Q116H mutants. The substrate profiles were similar for each mutant, but with significantly reduced activity compared with that of the wild-type. In contrast to the Q116H enzyme, which bound two zinc ions just like the wild-type, only one zinc ion is present in Q116A and Q116N. These results suggest that the Q116 residue plays a role in the binding of the zinc ion in the QHH site. [less ▲]

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See detailEffects of monopropanediamino-beta-cyclodextrin on the denaturation process of the hybrid protein BlaPChBD.
Vandevenne, Marylène ULg; GASPARD, Genevieve ULg; Belgsir, E. M. et al

in Biochimica et biophysica acta (2011)

Irreversible accumulation of protein aggregates represents an important problem both in vivo and in vitro. The aggregation of proteins is of critical importance in a wide variety of biomedical situations ... [more ▼]

Irreversible accumulation of protein aggregates represents an important problem both in vivo and in vitro. The aggregation of proteins is of critical importance in a wide variety of biomedical situations, ranging from diseases (such as Alzheimer's and Parkinson's diseases) to the production (e.g. inclusion bodies), stability, storage and delivery of protein drugs. beta-Cyclodextrin (beta-CD) is a circular heptasaccharide characterized by a hydrophilic exterior and a hydrophobic interior ring structure. In this research, we studied the effects of a chemically modified beta-CD (BCD07056), on the aggregating and refolding properties of BlaPChBD, a hybrid protein obtained by inserting the chitin binding domain of the human macrophage chitotriosidase into the class A beta-lactamase BlaP from Bacillus licheniformis 749/I during its thermal denaturation. The results show that BCD07056 strongly increases the refolding yield of BlaPChBD after thermal denaturation and constitutes an excellent additive to stabilize the protein over time at room temperature. Our data suggest that BCD07056 acts early in the denaturation process by preventing the formation of an intermediate which leads to an aggregated state. Finally, the role of beta-CD derivatives on the stability of proteins is discussed. [less ▲]

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See detailDistant and new mutations in CTX-M-1 beta-lactamase affect cefotaxime hydrolysis.
Perez-Llarena, Francisco J; Kerff, Frédéric ULg; Abian, Olga et al

in Antimicrobial Agents and Chemotherapy (2011), 55(9), 4361-8

The CTX-M beta-lactamases are an increasingly prevalent group of extended-spectrum beta-lactamases (ESBL). Point mutations in CTX-M beta-lactamases are considered critical for enhanced hydrolysis of ... [more ▼]

The CTX-M beta-lactamases are an increasingly prevalent group of extended-spectrum beta-lactamases (ESBL). Point mutations in CTX-M beta-lactamases are considered critical for enhanced hydrolysis of cefotaxime. In order to clarify the structural determinants of the activity against cefotaxime in CTX-M beta-lactamases, screening for random mutations was carried out to search for decreased activity against cefotaxime, with the CTX-M-1 gene as a model. Thirteen single mutants with a considerable reduction in cefotaxime MICs were selected for biochemical and stability studies. The 13 mutated genes of the CTX-M-1 beta-lactamase were expressed, and the proteins were purified for kinetic studies against cephalothin and cefotaxime (as the main antibiotics). Some of the positions, such as Val103Asp, Asn104Asp, Asn106Lys, and Pro107Ser, are located in the (103)VNYN(106) loop, which had been described as important in cefotaxime hydrolysis, although this has not been experimentally confirmed. There are four mutations located close to catalytic residues-Thr71Ile, Met135Ile, Arg164His, and Asn244Asp-that may affect the positioning of these residues. We show here that some distant mutations, such as Ala219Val, are critical for cefotaxime hydrolysis and highlight the role of this loop at the top of the active site. Other distant substitutions, such as Val80Ala, Arg191, Ala247Ser, and Val260Leu, are in hydrophobic cores and may affect the dynamics and flexibility of the enzyme. We describe here, in conclusion, new residues involved in cefotaxime hydrolysis in CTX-M beta-lactamases, five of which are in positions distant from the catalytic center. [less ▲]

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See detailCellulase involvement in the cellulose biosynthesis of Pseudomonas stutzeri
Delsaute, Maud ULg; Berlemont, Renaud ULg; Paulus, Virginie et al

Poster (2011)

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See detailExploring the Antarctic soil metagenome as a source of novel cold-adapted enzymes and genetic mobile elements
Berlemont, Renaud ULg; Pipers; Delsaute, Maud ULg et al

in Revista Argentina de Microbiologia (2011)

Metagenomic library PP1 was obtained from Antarctic soil samples. Both functional and genotypic metagenomic screening were used for the isolation of novel cold-adapted enzymes with potential applications ... [more ▼]

Metagenomic library PP1 was obtained from Antarctic soil samples. Both functional and genotypic metagenomic screening were used for the isolation of novel cold-adapted enzymes with potential applications, and for the detection of genetic elements associated with gene mobilization, respectively. Fourteen lipase/esterase-, 14 amylase-, 3 protease-, and 11 cellulase-producing clones were detected by activity-driven screening, with apparent maximum activities around 35 °C for both amylolytic and lipolytic enzymes, and 35-55 °C for cellulases, as observed for other cold-adapted enzymes. However, the behavior of at least one of the studied cellulases is more compatible to that observed for mesophilic enzymes. These enzymes are usually still active at temperatures above 60 °C, probably resulting in a psychrotolerant behavior in Antarctic soils. Metagenomics allows to access novel genes encoding for enzymatic and biophysic properties from almost every environment with potential benefits for biotechnological and industrial applications. Only intI- and tnp-like genes were detected by PC R, encoding for proteins with 58-86%, and 58-73% amino acid identity with known entries, respectively. Two clones, BAC 27A-9 and BAC 14A-5, seem to present unique syntenic organizations, suggesting the occurrence of gene rearrangements that were probably due to evolutionary divergences within the genus or facilitated by the association with transposable elements. The evidence for genetic elements related to recruitment and mobilization of genes (transposons/integrons) in an extreme environment like Antarctica reinforces the hypothesis [less ▲]

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See detailIndol-2-yl ethanones as novel indoleamine 2,3-dioxygenase (IDO) inhibitors.
Dolusic, Eduard; Larrieu, Pierre; Blanc, Sébastien et al

in Bioorganic & Medicinal Chemistry (2011), 19(4), 1550-61

Indoleamine 2,3-dioxygenase (IDO) is a heme dioxygenase which has been shown to be involved in the pathological immune escape of diseases such as cancer. The synthesis and structure-activity relationships ... [more ▼]

Indoleamine 2,3-dioxygenase (IDO) is a heme dioxygenase which has been shown to be involved in the pathological immune escape of diseases such as cancer. The synthesis and structure-activity relationships (SAR) of a novel series of IDO inhibitors based on the indol-2-yl ethanone scaffold is described. In vitro and in vivo biological activities have been evaluated, leading to compounds with IC(50) values in the micromolar range in both tests. Introduction of small substituents in the 5- and 6-positions of the indole ring, indole N-methylation and variations of the aromatic side chain are all well tolerated. An iron coordinating group on the linker is a prerequisite for biological activity, thus corroborating the virtual screening results. [less ▲]

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See detailDiscovery and preliminary SARs of keto-indoles as novel indoleamine 2,3-dioxygenase (IDO) inhibitors.
Dolusic, Eduard; Larrieu, Pierre; Blanc, Sebastien et al

in European journal of medicinal chemistry (2011), 46(7), 3058-65

Indoleamine 2,3-dioxygenase (IDO) is an important new therapeutic target for the treatment of cancer. With the aim of discovering novel IDO inhibitors, a virtual screen was undertaken and led to the ... [more ▼]

Indoleamine 2,3-dioxygenase (IDO) is an important new therapeutic target for the treatment of cancer. With the aim of discovering novel IDO inhibitors, a virtual screen was undertaken and led to the discovery of the keto-indole derivative 1a endowed with an inhibitory potency in the micromolar range. Detailed kinetics were performed and revealed an uncompetitive inhibition profile. Preliminary SARs were drawn in this series and corroborated the putative binding orientation as suggested by docking. [less ▲]

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