References of "Kerff, Frédéric"
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See detailOn the LZ distance for dereplicating redundant prokaryotic genomes
Léonard, Raphaël ULg; Baurain, Denis ULg; Kerff, Frédéric ULg et al

Poster (2015, December 07)

The fast-growing number of available prokaryotic genomes, along with their uneven taxonomic distribution, is a prob- lem when trying to assemble broadly sampled genome sets for phylogenomics and ... [more ▼]

The fast-growing number of available prokaryotic genomes, along with their uneven taxonomic distribution, is a prob- lem when trying to assemble broadly sampled genome sets for phylogenomics and comparative genomics. Indeed, most of the new genomes belong to the same subset of hyper-sampled phyla, such as Proteobacteria and Firmicutes, or even to single species, such as Escherichia coli (almost 2000 genomes as of Sept 2015), while the continuous flow of newly discovered phyla prompts for regular updates. This situation makes it difficult to maintain sets of representative genomes combining lesser known phyla, for which only few species are available, and sound subsets of highly abundant phyla. An automated straightforward method is required but none are publicly available. The LZ distance, in conjunction with the quality of the annotations, can be used to create an automated approach for selecting a subset of represen- tative genomes without redundancy. We are planning to release this tool on a website that will be made publicly available. [less ▲]

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See detailSPECIFICITY OF CLASS I TAGATOSE 1,6-BISPHOSPHATE ALDOLASE ENHANCED TOWARD TAGATOSE 1,6-BISPHOPHATE
Freichels, Régine ULg; Delmarcelle, Michaël ULg; Colarusso, Andrea et al

Poster (2015, July)

Class I tagatose 1,6-bisphosphate aldolase catalyzes the reversible condensation of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate to produce four D-ketohexoses 1,6-bisphosphate: D-tagatose 1,6 ... [more ▼]

Class I tagatose 1,6-bisphosphate aldolase catalyzes the reversible condensation of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate to produce four D-ketohexoses 1,6-bisphosphate: D-tagatose 1,6-bisphosphate, D-fructose 1,6-bisphosphate, D-psicose 1,6-bisphosphate and D-sorbose 1,6-bisphosphate. These four sugars are diastereoisomers and differs from each other in their stereochemistry at carbons 3 and 4. The structure determination of three class I tagatose 1,6-bisphosphate aldolases has afforded new insight into their catalytic mechanism as well as their evolution. However, the determinant(s) that allow(s) the enzyme to be so unspecific at carbon 4 have remains unknown. The aim of this project is focused on the characterization of the structural features of tagatose 1,6-bisphosphate aldolases that determine the specificity of the enzyme towards tagatose versus fructose 1,6-bisphosphate (carbon C4). [less ▲]

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See detailCharacterization of YbjG, a pyrophosphate phosphatase from E. coli involved in the lipid carrier undecaprenyl phosphate metabolism
Delbrassine, François ULg; Auger, Rodolphe; El Ghachi, Meriem ULg et al

Poster (2015, June 08)

•Background Undecaprenyl phosphate (C55-P) is an essential lipid carrier involved in the biosynthesis of cell surface carbohydrate polymers such as the peptidoglycan. C55-P is the result of the ... [more ▼]

•Background Undecaprenyl phosphate (C55-P) is an essential lipid carrier involved in the biosynthesis of cell surface carbohydrate polymers such as the peptidoglycan. C55-P is the result of the dephosphorylation of the undecaprenyl pyrophosphate (C55-PP) by specific phosphatases. In Escherichia coli this dephosphorylation can be performed by four integral membrane proteins, BacA, and three members of the type 2 phosphatidic acid phosphatase family (PAP2), PgpB, YbjG, and LpxT. •Objectives The aim of this project is to characterize YbjG and contributes to the understanding of the physiological role and mechanism of action of this enzyme in the C55-P metabolism. The C55-PP phosphatases could become an interesting target in the search for new molecules with antibacterial activity. •Methods In parallel the stability of YbjG and its activity against C15-PP were assessed in 94 different detergents. Moreover the enzymatic activity of YbjG was studied: substrate specificity, optimum pH and temperature, effect of detergent concentration. •Conclusions For the first time, YbjG has been purified and we show its ability to dephosphorylate C15-PP, DGPP and C55-PP in vitro with respectively decreasing efficiency. No activity has been detected on five other potential substrates (PPi, PA, C5-PP, G6P & PNPP). The phosphatase activity on C15-PP is maximum at pH 6,5 and 25 °C. Moreover Cymal6, LMNG, & ωUDM are good detergent both for the stability and the C15-PP phosphatase activity of YbjG, but approximately half of the 94 tested detergents show C15-PP phosphatase activity on the qualitative enzymatic test. [less ▲]

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See detailDeciphering the metabolism of polyprenyl-phosphate: The universal glycan lipid carrier at the membrane frontier
Manat, Guillaume; Roure, Sophie; El Ghachi, Meriem ULg et al

Poster (2015, June)

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See detailRésistance aux antibiotiques : le retour à l’ère prébiotique ?
Galleni, Moreno ULg; Kerff, Frédéric ULg; Rigali, Sébastien ULg

Conference (2015, February 05)

Aujourd’hui, la résistance des bactéries aux antibiotiques relève d'une préoccupation croissante dans le domaine de la santé publique et s'impose plus que jamais comme une priorité. Ce problème a un coût ... [more ▼]

Aujourd’hui, la résistance des bactéries aux antibiotiques relève d'une préoccupation croissante dans le domaine de la santé publique et s'impose plus que jamais comme une priorité. Ce problème a un coût, sociétal et économique en Europe: 25.000 décès annuels par septicémie et 1,5 milliard d’euros d’augmentation du coût des traitements. Bien que le besoin de nouveaux agents antibactériens en milieu clinique est urgent, seules deux nouvelles classes d’antibiotiques ont pu être proposées durant ces 30 dernières années. En effet, la découverte et le développement de nouveaux antibiotiques posent des défis scientifiques, cliniques et financiers importants. [less ▲]

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See detailStructural and Kinetic Insights into the "Ceftazidimase" Behavior of the Extended-Spectrum beta-Lactamase CTX-M-96.
Ghiglione, Barbara; Rodriguez, Maria Margarita; Herman, Raphaël ULg et al

in Biochemistry (2015), 54(32), 5072-82

Diversification of the CTX-M beta-lactamases led to the emergence of variants responsible for decreased susceptibility to ceftazidime, like the Asp240Gly-harboring "ceftazidimases". We solved the ... [more ▼]

Diversification of the CTX-M beta-lactamases led to the emergence of variants responsible for decreased susceptibility to ceftazidime, like the Asp240Gly-harboring "ceftazidimases". We solved the crystallographic structure of the Asp240Gly variant CTX-M-96 at 1.2 A and evaluated the role of Asp240 in the activity toward oxyimino-cephalosporins through simulated models and kinetics. There seem to be subtle changes in the conformation of the active site cavity of CTX-M-96, compared to enzyme variants harboring the Asp240, and these small rearrangements could be due to localized shifts in the environment of the beta3 strand. According to the crystallographic evidence, CTX-M-96 presents a "compact" active site, which in spite of its reduced cavity seems to allow the proper interaction with oxyimino-cephalosporins, as suggested by simulated models. The term "ceftazidimases" that is currently applied for the Asp240Gly-harboring CTX-M variants should be used carefully. Structural differences between CTX-M harboring the Asp240Gly mutation (and also probably others like those at Pro167) do not seem to be conclusive to determine the "ceftazidimase" behavior observed in vivo, which is in turn partially supported by the mild improvement in the catalytic efficiency toward ceftazidime by CTX-M-96 and similar enzymes, compared to "parental" Asp240-harboring variants. In addition, it is observed that alterations in OmpF expression could act synergistically with CTX-M-96 for yielding clinical resistance toward ceftazidime. We therefore propose that the observed resistance in vivo is due to the sum of synergic mechanisms, and the term "cefotaximases associated with ceftazidime resistance" could be conveniently used to describe CTX-M harboring the Asp240Gly substitution. [less ▲]

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See detailMembrane Topology and Biochemical Characterization of the Escherichia coli BacA Undecaprenyl-Pyrophosphate Phosphatase.
Manat, Guillaume; El Ghachi, Meriem ULg; Auger, Rodolphe et al

in PloS one (2015), 10(11), 0142870

Several integral membrane proteins exhibiting undecaprenyl-pyrophosphate (C55-PP) phosphatase activity were previously identified in Escherichia coli that belonged to two distinct protein families: the ... [more ▼]

Several integral membrane proteins exhibiting undecaprenyl-pyrophosphate (C55-PP) phosphatase activity were previously identified in Escherichia coli that belonged to two distinct protein families: the BacA protein, which accounts for 75% of the C55-PP phosphatase activity detected in E. coli cell membranes, and three members of the PAP2 phosphatidic acid phosphatase family, namely PgpB, YbjG and LpxT. This dephosphorylation step is required to provide the C55-P carrier lipid which plays a central role in the biosynthesis of various cell wall polymers. We here report detailed investigations of the biochemical properties and membrane topology of the BacA protein. Optimal activity conditions were determined and a narrow-range substrate specificity with a clear preference for C55-PP was observed for this enzyme. Alignments of BacA protein sequences revealed two particularly well-conserved regions and several invariant residues whose role in enzyme activity was questioned by using a site-directed mutagenesis approach and complementary in vitro and in vivo activity assays. Three essential residues Glu21, Ser27, and Arg174 were identified, allowing us to propose a catalytic mechanism for this enzyme. The membrane topology of the BacA protein determined here experimentally did not validate previous program-based predicted models. It comprises seven transmembrane segments and contains in particular two large periplasmic loops carrying the highly-conserved active site residues. Our data thus provide evidence that all the different E. coli C55-PP phosphatases identified to date (BacA and PAP2) catalyze the dephosphorylation of C55-PP molecules on the same (outer) side of the plasma membrane. [less ▲]

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See detailFrom << An enzyme able to destroy penicillin >> to carbapenemases: 70 years of beta-lactamase misbehavior.
Frère, Jean-Marie ULg; Sauvage, Eric ULg; Kerff, Frederic ULg

in Current drug targets (2015)

As early as 1940, Abraham and Chain described "an enzyme able to destroy penicillin". In the late 1940's, penicillin-resistant strains of Staphylococcus aureus were found to be a clinical problem. They ... [more ▼]

As early as 1940, Abraham and Chain described "an enzyme able to destroy penicillin". In the late 1940's, penicillin-resistant strains of Staphylococcus aureus were found to be a clinical problem. They produced a penicillinase that could hydrolyze the amide bond in the beta-lactam ring. Later, an enzyme mediated by an R-factor was isolated from Enterobacteriaceae. Methicillin and cephalosporins, both very poor substrates of the S. aureus enzyme, were found to be sensitive to this new enzyme. Third generation cephalosporins appeared to solve the problem, but further enzymes were selected that exhibited extended spectra and could for instance hydrolyze cefotaxime and/or ceftazidime. The discovery of carbapenems constituted a major advance for our antimicrobial arsenal: they inactivated most of the essential penicillin binding proteins effectively and escaped the activity of nearly all known beta-lactamases. However, the metallo-beta-lactamases, which had not been recognised as a major danger before 1990, were found to act as effective carbapenemases and started to spread in a worrying way. Moreover, carbapenem-hydrolyzing enzymes were found in each of the 3 classes of active-site serine beta-lactamases. [less ▲]

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See detailOverview of the synthesis and structural studies of class D β-lactamases inhibitor
Kerff, Frédéric ULg; Caroen, Jurgen

Conference (2014, April 23)

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See detailCrystal structure of penicillin-binding protein 3 (PBP3) from Escherichia coli
Sauvage, Eric ULg; Derouaux, Adeline ULg; Fraipont, Claudine ULg et al

in PLoS ONE (2014)

In Escherichia coli, penicillin-binding protein 3 (PBP3), also known as FtsI, is a central component of the divisome, catalyzing cross-linking of the cell wall peptidoglycan during cell division. PBP3 is ... [more ▼]

In Escherichia coli, penicillin-binding protein 3 (PBP3), also known as FtsI, is a central component of the divisome, catalyzing cross-linking of the cell wall peptidoglycan during cell division. PBP3 is mainly periplasmic, with a 23 residues cytoplasmic tail and a single transmembrane helix. We have solved the crystal structure of a soluble form of PBP3 (PBP357-577) at 2.5 Å revealing the two modules of high molecular weight class B PBPs, a carboxy terminal module exhibiting transpeptidase activity and an amino terminal module with unknown function. To gain additional insight, the PBP3 Val88-Ser165 subdomain (PBP388-165), for which the electron density is poorly defined in the PBP3 crystal, was produced and its structure solved by SAD phasing at 2.1 Å. The structure shows a three dimensional domain swapping with a β-strand of one molecule inserted between two strands of the paired molecule, suggesting a possible role in PBP357-577 dimerization. [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 detailThe cell separation step of bacterial cell division
Kerff, Frédéric ULg

Scientific conference (2013, November 26)

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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 detailCharacterization of the new AmpC beta-lactamase FOX-8 reveals a single mutation, Phe313Leu, located in the R2 loop that affects ceftazidime hydrolysis.
Perez-Llarena, Francisco Jose; Kerff, Frédéric ULg; Zamorano, Laura et al

in Antimicrobial agents and chemotherapy (2013), 57(10), 5158-61

A novel class C beta-lactamase (FOX-8) was isolated from a clinical strain of Escherichia coli. The FOX-8 enzyme possessed a unique substitution (Phe313Leu) compared to FOX-3. Isogenic E. coli strains ... [more ▼]

A novel class C beta-lactamase (FOX-8) was isolated from a clinical strain of Escherichia coli. The FOX-8 enzyme possessed a unique substitution (Phe313Leu) compared to FOX-3. Isogenic E. coli strains carrying FOX-8 showed an 8-fold reduction in resistance to ceftazidime relative to FOX-3. In a kinetic analysis, FOX-8 displayed a 33-fold reduction in kcat/Km for ceftazidime compared to FOX-3. In the FOX family of beta-lactamases, the Phe313 residue located in the R2 loop affects ceftazidime hydrolysis and alters the phenotype of E. coli strains carrying this variant. [less ▲]

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