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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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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 ULg 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 detailBiochemical and structural characterization of the subclass B1 metallo-β-lactamase VIM-4.
Lassaux, Patricia ULg; Traoré, Daouda; Loisel, Elodie et al

in Antimicrobial Agents and Chemotherapy (2011)

The metallo-β-lactamase VIM-4, mainly found in Pseudomonas aeruginosa or Acinetobacter baumannii, was produced in Escherichia coli and characterized by biochemical and X-ray techniques. A detailed kinetic ... [more ▼]

The metallo-β-lactamase VIM-4, mainly found in Pseudomonas aeruginosa or Acinetobacter baumannii, was produced in Escherichia coli and characterized by biochemical and X-ray techniques. A detailed kinetic study performed in the presence of Zn²+ at concentrations ranging from 0.4 to 100 μM showed that VIM-4 exhibits a kinetic profile similar to the profiles of VIM-2 and VIM-1. However, VIM-4 is more active than VIM-1 against benzylpenicillin, cephalothin, nitrocefin, and imipenem and is less active than VIM-2 against ampicillin and meropenem. The crystal structure of the dizinc form of VIM-4 was solved at 1.9 Å. The sole difference between VIM-4 and VIM-1 is found at residue 228, which is Ser in VIM-1 and Arg in VIM-4. This substitution has a major impact on the VIM-4 catalytic efficiency compared to that of VIM-1. In contrast, the differences between VIM-2 and VIM-4 seem to be due to a different position of the flapping loop and two substitutions in loop 2. Study of the thermal stability and the activity of the holo- and apo-VIM-4 enzymes revealed that Zn²+ ions have a pronounced stabilizing effect on the enzyme and are necessary for preserving the structure. [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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