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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 detailCrystal structure of penicillin-binding protein 3 (PBP3) from Escherichia coli
Sauvage, Eric; 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 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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See detailThe crystal structure of the cell division amidase AmiC reveals the fold of the AMIN domain, a new peptidoglycan binding domain.
Rocaboy, Mathieu; Herman, Raphael; Sauvage, Eric ULg et al

in Molecular microbiology (2013)

Binary fission is the ultimate step of the prokaryotic cell cycle. In Gram-negative bacteria like Escherichia coli, this step implies the invagination of three biological layers (cytoplasmic membrane ... [more ▼]

Binary fission is the ultimate step of the prokaryotic cell cycle. In Gram-negative bacteria like Escherichia coli, this step implies the invagination of three biological layers (cytoplasmic membrane, peptidoglycan and outer membrane), biosynthesis of the new poles and eventually, daughter cells separation. The latter requires the coordinated action of the N-acetylmuramyl-L-alanine amidases AmiA/B/C and their LytM activators EnvC and NlpD to cleave the septal peptidoglycan. We present here the 2.5 A crystal structure of AmiC which includes the first report of an AMIN domain structure, a beta-sandwich of two symmetrical four-stranded beta-sheets exposing highly conserved motifs on the two outer faces. We show that this N-terminal domain, involved in the localization of AmiC at the division site, is a new peptidoglycan-binding domain. The C-terminal catalytic domain shows an auto-inhibitory alpha helix obstructing the active site. AmiC lacking this helix exhibits by itself an activity comparable to that of the wild type AmiC activated by NlpD. We also demonstrate the interaction between AmiC and NlpD by microscale thermophoresis and confirm the importance of the active site blocking alpha helix in the regulation of the amidase activity. [less ▲]

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See detailInhibition of dd-Peptidases by a Specific Trifluoroketone: Crystal Structure of a Complex with the Actinomadura R39 dd-Peptidase.
Dzhekieva, Liudmila; Adediran, S. A.; Herman, Raphael et al

in Biochemistry (2013)

Inhibitors of bacterial dd-peptidases represent potential antibiotics. In the search for alternatives to beta-lactams, we have investigated a series of compounds designed to generate transition state ... [more ▼]

Inhibitors of bacterial dd-peptidases represent potential antibiotics. In the search for alternatives to beta-lactams, we have investigated a series of compounds designed to generate transition state analogue structures upon reaction with dd-peptidases. The compounds contain a combination of a peptidoglycan-mimetic specificity handle and a warhead capable of delivering a tetrahedral anion to the enzyme active site. The latter includes a boronic acid, two alcohols, an aldehyde, and a trifluoroketone. The compounds were tested against two low-molecular mass class C dd-peptidases. As expected from previous observations, the boronic acid was a potent inhibitor, but rather unexpectedly from precedent, the trifluoroketone [d-alpha-aminopimelyl(1,1,1-trifluoro-3-amino)butan-2-one] was also very effective. Taking into account competing hydration, we found the trifluoroketone was the strongest inhibitor of the Actinomadura R39 dd-peptidase, with a subnanomolar (free ketone) inhibition constant. A crystal structure of the complex between the trifluoroketone and the R39 enzyme showed that a tetrahedral adduct had indeed formed with the active site serine nucleophile. The trifluoroketone moiety, therefore, should be considered along with boronic acids and phosphonates as a warhead that can be incorporated into new and effective dd-peptidase inhibitors and therefore, perhaps, antibiotics. [less ▲]

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See detailStructural Determinants of Specificity and Catalytic Mechanism in mammalian 25-kDa Thiamine Triphosphatase
Delvaux, David; Kerff, Frédéric ULg; Murty, Mamidanna R.V.S. et al

in Biochimica et Biophysica Acta - General Subjects (2013), 1830

Background: Thiamine triphosphate (ThTP) is present in most organisms and might be involved in intracellular signaling. In mammalian cells, the cytosolic ThTP level is controlled by a specific thiamine ... [more ▼]

Background: Thiamine triphosphate (ThTP) is present in most organisms and might be involved in intracellular signaling. In mammalian cells, the cytosolic ThTP level is controlled by a specific thiamine triphosphatase (ThTPase), belonging to the CYTH superfamily of proteins. CYTH proteins are present in all superkingdoms of life and act on various triphosphorylated substrates. Methods: Using crystallography, mass spectrometry and mutational analysis, we identified the key structural determinants of the high specificity and catalytic efficiency of mammalian ThTPase. Results: Triphosphate binding requires three conserved arginines while the catalytic mechanism relies on an unusual lysine-tyrosine dyad. By docking of the ThTP molecule in the active site, we found that Trp-53 should interact with the thiazole part of the substrate molecule, thus playing a key role in substrate recognition and specificity. Sea anemone and zebrafish CYTH proteins, which retain the corresponding Trp residue, are also specific ThTPases. Surprisingly, the whole chromosome region containing the ThTPase gene is lost in birds. Conclusion: The specificity for ThTP is linked to a stacking interaction between the thiazole heterocycle of thiamine and a tryptophan residue. The latter likely plays a key role in the secondary acquisition of ThTPase activity in early metazoan CYTH enzymes, in the lineage leading from cnidarians to mammals. General significance: We show that ThTPase activity is not restricted to mammals as previously thought but is an acquisition of early metazoans. This, and the identification of critically important residues, allows us to draw an evolutionary perspective of the CYTH family of proteins. [less ▲]

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See detailCharacterization of a novel IMP-28 metallo-β-lactamase from a Spanish Klebsiella oxytoca clinical isolate
Pérez-Llarena, FJ; Fernández, A; Zamorano, L et al

in Antimicrobial Agents and Chemotherapy (2012), 56(8), 4540-3

An isolate of Klebsiella oxytoca carrying a novel IMP metallo-β-lactamase was discovered in Madrid, Spain. The bla(IMP-28) gene is part of a chromosomally located class I integron. The IMP-28 k(cat)/K(m ... [more ▼]

An isolate of Klebsiella oxytoca carrying a novel IMP metallo-β-lactamase was discovered in Madrid, Spain. The bla(IMP-28) gene is part of a chromosomally located class I integron. The IMP-28 k(cat)/K(m) values for ampicillin, ceftazidime, and cefepime and, to a lesser extent, imipenem and meropenem, are clearly lower than those of IMP-1. The His306Gln mutation may induce important modifications of the L3 loop and thus of substrate accessibility and hydrolysis and be the main reason for this behavior. [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 detailUnexpected tricovalent binding mode of boronic acids within the active site of a penicillin binding protein.
Zervosen, Astrid ULg; Herman, Raphaël ULg; Kerff, Frédéric ULg et al

in Journal of the American Chemical Society (2011)

Boronic acids bearing appropriate side chains are good inhibitors of serine amidohydrolases. The boron usually adopts a tetrahedral conformation, bound to the nucleophilic serine of the active site and ... [more ▼]

Boronic acids bearing appropriate side chains are good inhibitors of serine amidohydrolases. The boron usually adopts a tetrahedral conformation, bound to the nucleophilic serine of the active site and mimicking the transition state of the enzymatic reaction. We have solved the structures of complexes of a penicillin-binding protein, the DD-peptidase from Actinomadura sp. R39, with four amidomethylboronic acids (2,6 dimethoxybenzamidomethylboronic acid, phenylacetamidomethylboronic acid, 2-chlorobenzamidomethylboronic acid, and 2-nitrobenzamidomethylboronic acid) and the pinacol ester derived from phenylacetamidomethylboronic acid. We found that, in each case, the boron forms a tricovalent adduct with Ogamma of Ser49, Ser298, and the terminal amine group of Lys410, three key residues involved in the catalytic mechanism of penicillin-binding proteins. This represents the first tricovalent enzyme-inhibitor adducts observed by crystallography. In two of the five R39-boronate structures, the boronic acid is found as a tricovalent adduct in two monomers of the asymmetric unit and as a monocovalent adduct with the active serine in the two remaining monomers of the asymmetric unit. Formation of the tricovalent complex from a classical monocovalent complex may involve rotation around the Ser49 Calpha-Cbeta bond to place the boron in a position to interact with Ser298 and Lys410, and a twisting of the side chain amide such that its carbonyl oxygen is able to hydrogen bond to the oxyanion hole NH of Thr413. Biphasic kinetics were observed in three of the five cases and details of the reaction between R39 and 2,6-dimethoxybenzamidomethylboronic acid were studied. Observation of biphasic kinetics was not, however, thought to be correlated to formation of tricovalent complexes, assuming that the latter do form in solution. Based on the crystallographic and kinetic results, a reaction scheme for this unexpected inhibition by boronic acids is proposed. [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 detailStructures of class D β-lactamases
Kerff, Frédéric ULg; Sauvage, Eric ULg; Vercheval, Lionel ULg et al

in Frère, Jean-Marie (Ed.) Beta-lactamases (2011)

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See detailThree factors that modulate the activity of class D β-lactamases and interfere with the post-translational carboxylation of Lys 70
Vercheval, Lionel ULg; Di Paolo, Alexandre ULg; Borel, Franck et al

in Biochemical Journal (2010), 432(3), 495-504

Lys-70 carboxylation in the active site of class D β lactamases is essential for their activity. Structural, kinetic and affinity studies show that this post-translational modification can be affected by ... [more ▼]

Lys-70 carboxylation in the active site of class D β lactamases is essential for their activity. Structural, kinetic and affinity studies show that this post-translational modification can be affected by the presence of a poor substrate such as moxalactam but also by the V117T substitution. Val-117 is a strictly conserved hydrophobic residue located in the active site. In addition, inhibition of class D β lactamases by chloride ions is due to a competition between the side chain carboxylate of the modified Lys 70 and chloride ions. Determination of the individual kinetic constants shows that the deacylation of the acyl-enzyme is the rate limiting step for the wild type OXA 10 β lactamase. [less ▲]

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See detailA Tripeptide Deletion in the Class C beta-Lactamase FOX-4 Enzyme Impairs Cefoxitin Hydrolysis and Slightly Increases Susceptibility to beta-Lactamase Inhibitors
Mallo, Susana; Pérez-Llarena, Francisco J.; Kerff, Frédéric ULg et al

in Journal of Antimicrobial Chemotherapy (2010), 65(6), 1187-94

OBJECTIVES: A natural variant of the AmpC enzyme from Escherichia coli HKY28 with a tripeptide deletion (Gly-286/Ser-287/Asp-288) was recently described. The isolate produced an inhibitor-sensitive AmpC ... [more ▼]

OBJECTIVES: A natural variant of the AmpC enzyme from Escherichia coli HKY28 with a tripeptide deletion (Gly-286/Ser-287/Asp-288) was recently described. The isolate produced an inhibitor-sensitive AmpC beta-lactamase variant that also conferred higher than usual levels of resistance to ceftazidime in the E. coli host. To demonstrate whether this is true in other class C beta-lactamase enzymes, we deleted the equivalent tripeptide in the FOX-4 plasmid-mediated class C beta-lactamase. METHODS: By site-directed mutagenesis, we deleted the tripeptide Gly-306/Asn-307/Ser-308 of FOX-4, thus generating FOX-4(DeltaGNS). The enzymes (FOX-4 wild-type and DeltaGNS) were purified and kinetic parameters (kcat, Km, kcat/Km) as well as IC50 values of several beta-lactams were assessed. Modelling studies were also performed. RESULTS: FOX-4(DeltaGNS) did not increase the catalytic efficiency towards ceftazidime, although it conferred a slight increase in the susceptibility to beta-lactamase inhibitors. There was also a noteworthy decrease in the cefoxitin MIC with the FOX-4(DeltaGNS) mutant (from 512 to 16 mg/L) as well as a 10-fold decrease in kcat/Km towards imipenem, which revealed specific structural features. CONCLUSIONS: Although deletions in the R2-loop are able to extend the substrate spectrum of class C enzymes, the present results do not confirm this hypothesis in FOX-4. The FOX-4 R2 site would already be wide enough to accommodate antibiotic molecules, and thus any amino acid replacement or deletion at this location would not affect the hydrolytic efficiency towards beta-lactams and would have a less drastic effect on the susceptibility to beta-lactamase inhibitors. [less ▲]

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