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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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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 detailHow to make good use of a “bad” enzyme: utilization of efficient β-lactamases for the benefits of biochemical research
Vandevenne, Marylène ULg; Galleni, Moreno ULg; Filée, Patrice

in Frère, Jean-Marie (Ed.) β-lactamases (2012)

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See detailSynthesis, crystal structures and electronic properties of isomers of chloro-pyridinylvinyl-1H-indoles.
Moineaux, Laurence; Laurent, Sophie; Reniers, Jeremy et al

in European journal of medicinal chemistry (2012), 54

Three isomers of chloro-3-(2-pyridin-3-ylvinyl)-1H-indole were synthesized and tested as inhibitors of human tryptophan 2,3-dioxygenase (hTDO). The crystal structures of two of them were solved by X-ray ... [more ▼]

Three isomers of chloro-3-(2-pyridin-3-ylvinyl)-1H-indole were synthesized and tested as inhibitors of human tryptophan 2,3-dioxygenase (hTDO). The crystal structures of two of them were solved by X-ray diffraction. The solubility of the molecules also was determined experimentally. The molecular electrostatic potentials and dipole moments of the three isomers were calculated by ab initio quantum mechanics (HF/6-311G). The single crystal X-ray analyses reveal non-planar structures. This non-coplanarity is retained during docking of the compounds into a model of hTDO, the molecular target of this series. The position of the Cl atom does not significantly affect the electronic delocalization. Nevertheless, the position of the Cl atom produces a local variation of bond lengths inducing different dipole moments for these isomers. Variations in dipole moments are consistent with the different melting points and crystal packings. Differences in aqueous solubilities are best explained by subtle changes in H-bonds resulting from different accessibilities of the indole NH's due to steric effects of the Cl substituent. The non-coplanarity plays an important role in the crystalline packing of the molecules in contrast to the position of the Cl. This study leads to a better understanding of the structural and electronic characteristics of this chemical series and can potentially help to better understand their inhibitory activity. [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 detailComparative functional analysis of the human macrophage chitotriosidase
Vandevenne, Marylène ULg; Campisi, Vincenzo ULg; Freichels, Astrid ULg et al

in Protein Science : A Publication of the Protein Society (2011)

Detailed reference viewed: 43 (1 ULg)