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See detailCritical role of tryptophan 154 for the activity and stability of class D beta-lactamases.
Baurin, Stephane; Vercheval, Lionel ULg; Bouillenne, Fabrice ULg et al

in Biochemistry (2009), 48(47), 11252-63

The catalytic efficiency of the class D beta-lactamase OXA-10 depends critically on an unusual carboxylated lysine as the general base residue for both the enzyme acylation and deacylation steps of ... [more ▼]

The catalytic efficiency of the class D beta-lactamase OXA-10 depends critically on an unusual carboxylated lysine as the general base residue for both the enzyme acylation and deacylation steps of catalysis. Evidence is presented that the interaction between the indole group of Trp154 and the carboxylated lysine is essential for the stability of the posttranslationally modified Lys70. Substitution of Trp154 by Gly, Ala, or Phe yielded noncarboxylated enzymes which displayed poor catalytic efficiencies and reduced stability when compared to the wild-type OXA-10. The W154H mutant was partially carboxylated. In addition, the maximum values of k(cat) and k(cat)/K(M) were shifted toward pH 7, indicating that the carboxylation state of Lys70 is dependent on the protonation level of the histidine. A comparison of the three-dimensional structures of the different proteins also indicated that the Trp154 mutations did not modify the overall structures of OXA-10 but induced an increased flexibility of the Omega-loop in the active site. Finally, the deacylation-impaired W154A mutant was used to determine the structure of the acyl-enzyme complex with benzylpenicillin. These results indicate a role of the Lys70 carboxylation during the deacylation step and emphasize the importance of Trp154 for the ideal positioning of active site residues leading to an optimum activity. [less ▲]

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See detailThe Penicillin-Binding Proteins: Structure and Role in Peptidoglycan Biosynthesis
Sauvage, Eric ULg; Kerff, Frédéric ULg; Terrak, Mohammed ULg et al

in FEMS Microbiology Reviews (2008), 32(2), 234-58

Penicillin-binding proteins (PBPs) have been scrutinized for over 40 years. Recent structural information on PBPs together with the ongoing long-term biochemical experimental investigations, and results ... [more ▼]

Penicillin-binding proteins (PBPs) have been scrutinized for over 40 years. Recent structural information on PBPs together with the ongoing long-term biochemical experimental investigations, and results from more recent techniques such as protein localization by green fluorescent protein-fusion immunofluorescence or double-hybrid assay, have brought our understanding of the last stages of the peptidoglycan biosynthesis to an outstanding level that allows a broad outlook on the properties of these enzymes. Details are emerging regarding the interaction between the peptidoglycan-synthesizing PBPs and the peptidoglycan, their mesh net-like product that surrounds and protects bacteria. This review focuses on the detailed structure of PBPs and their implication in peptidoglycan synthesis, maturation and recycling. An overview of the content in PBPs of some bacteria is provided with an emphasis on comparing the biochemical properties of homologous PBPs (orthologues) belonging to different bacteria. [less ▲]

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See detailTem-1 Beta-Lactamase Folds in a Nonhierarchical Manner with Transient Non-Native Interactions Involving the C-Terminal Region
Lejeune, Annabelle ULg; Pain, R. H.; Charlier, Paulette ULg et al

in Biochemistry (2008), 47(4), 1186-93

The conformational stability and kinetics of refolding and unfolding of the W290F mutant of TEM-1 beta-lactamase have been determined as a function of guanidinium chloride concentration. The activity and ... [more ▼]

The conformational stability and kinetics of refolding and unfolding of the W290F mutant of TEM-1 beta-lactamase have been determined as a function of guanidinium chloride concentration. The activity and spectroscopic properties of the mutant enzyme did not differ significantly from those of the wild type, indicating that the mutation has only a very limited effect on the structure of the protein. The stability of the folded protein is reduced, however, by 5-10 kJ mol-1 relative to that of the molten globule intermediate (H), but the values of the folding rate constants are unchanged, suggesting that Trp-290 becomes organized in its nativelike environment only after the rate-limiting step; i.e., the C-terminal region of the enzyme folds very late. In contrast to the significant increase in fluorescence intensity seen in the dead time (3-4 ms) of refolding of the wild-type protein, no corresponding burst phase was observed with the mutant enzyme, enabling the burst phase to be attributed specifically to the C-terminal Trp-290. This residue is suggested to be buried in a nonpolar environment from which it has to escape during subsequent folding steps. With both proteins, fast early collapse leads to a folding intermediate in which the C-terminal region of the polypeptide chain is trapped in a non-native structure, consistent with a nonhierarchical folding process. [less ▲]

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See detailCrystal structure of a cold-adapted class C beta-lactamase.
Michaux, Catherine; Massant, Jan; Kerff, Frédéric ULg et al

in FEBS Journal (2008), 275(8), 1687-97

In this study, the crystal structure of a class C beta-lactamase from a psychrophilic organism, Pseudomonas fluorescens, has been refined to 2.2 A resolution. It is one of the few solved crystal ... [more ▼]

In this study, the crystal structure of a class C beta-lactamase from a psychrophilic organism, Pseudomonas fluorescens, has been refined to 2.2 A resolution. It is one of the few solved crystal structures of psychrophilic proteins. The structure was compared with those of homologous mesophilic enzymes and of another, modeled, psychrophilic protein. The elucidation of the 3D structure of this enzyme provides additional insights into the features involved in cold adaptation. Structure comparison of the psychrophilic and mesophilic beta-lactamases shows that electrostatics seems to play a major role in low-temperature adaptation, with a lower total number of ionic interactions for cold enzymes. The psychrophilic enzymes are also characterized by a decreased number of hydrogen bonds, a lower content of prolines, and a lower percentage of arginines in comparison with lysines. All these features make the structure more flexible so that the enzyme can behave as an efficient catalyst at low temperatures. [less ▲]

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See detailBacterial peptidoglycan (murein) hydrolases.
Vollmer, Waldemar; Joris, Bernard ULg; Charlier, Paulette ULg et al

in FEMS Microbiology Reviews (2008), 32(2), 259-86

Most bacteria have multiple peptidoglycan hydrolases capable of cleaving covalent bonds in peptidoglycan sacculi or its fragments. An overview of the different classes of peptidoglycan hydrolases and ... [more ▼]

Most bacteria have multiple peptidoglycan hydrolases capable of cleaving covalent bonds in peptidoglycan sacculi or its fragments. An overview of the different classes of peptidoglycan hydrolases and their cleavage sites is provided. The physiological functions of these enzymes include the regulation of cell wall growth, the turnover of peptidoglycan during growth, the separation of daughter cells during cell division and autolysis. Specialized hydrolases enlarge the pores in the peptidoglycan for the assembly of large trans-envelope complexes (pili, flagella, secretion systems), or they specifically cleave peptidoglycan during sporulation or spore germination. Moreover, peptidoglycan hydrolases are involved in lysis phenomena such as fratricide or developmental lysis occurring in bacterial populations. We will also review the current view on the regulation of autolysins and on the role of cytoplasm hydrolases in peptidoglycan recycling and induction of beta-lactamase. [less ▲]

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See detailCrystal structure and activity of Bacillus subtilis YoaJ (EXLX1), a bacterial expansin that promotes root colonization.
Kerff, Frédéric ULg; Amoroso, Ana Maria ULg; Herman, Raphaël ULg et al

in Proceedings of the National Academy of Sciences of the United States of America (2008), 105(44), 16876-81

We solved the crystal structure of a secreted protein, EXLX1, encoded by the yoaJ gene of Bacillus subtilis. Its structure is remarkably similar to that of plant beta-expansins (group 1 grass pollen ... [more ▼]

We solved the crystal structure of a secreted protein, EXLX1, encoded by the yoaJ gene of Bacillus subtilis. Its structure is remarkably similar to that of plant beta-expansins (group 1 grass pollen allergens), consisting of 2 tightly packed domains (D1, D2) with a potential polysaccharide-binding surface spanning the 2 domains. Domain D1 has a double-psi beta-barrel fold with partial conservation of the catalytic site found in family 45 glycosyl hydrolases and in the MltA family of lytic transglycosylases. Domain D2 has an Ig-like fold similar to group 2/3 grass pollen allergens, with structural features similar to a type A carbohydrate-binding domain. EXLX1 bound to plant cell walls, cellulose, and peptidoglycan, but it lacked lytic activity against a variety of plant cell wall polysaccharides and peptidoglycan. EXLX1 promoted plant cell wall extension similar to, but 10 times weaker than, plant beta-expansins, which synergistically enhanced EXLX1 activity. Deletion of the gene encoding EXLX1 did not affect growth or peptidoglycan composition of B. subtilis in liquid medium, but slowed lysis upon osmotic shock and greatly reduced the ability of the bacterium to colonize maize roots. The presence of EXLX1 homologs in a small but diverse set of plant pathogens further supports a role in plant-bacterial interactions. Because plant expansins have proved difficult to express in active form in heterologous systems, the discovery of a bacterial homolog opens the door for detailed structural studies of expansin function. [less ▲]

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See detailCrystal structures of complexes of bacterial DD-peptidases with peptidoglycan-mimetic ligands: the substrate specificity puzzle.
Sauvage, Eric ULg; Powell, Ailsa J; Heilemann, Jason et al

in Journal of Molecular Biology (2008), 381(2), 383-93

The X-ray crystal structures of covalent complexes of the Actinomadura R39 dd-peptidase and Escherichia coli penicillin-binding protein (PBP) 5 with beta-lactams bearing peptidoglycan-mimetic side chains ... [more ▼]

The X-ray crystal structures of covalent complexes of the Actinomadura R39 dd-peptidase and Escherichia coli penicillin-binding protein (PBP) 5 with beta-lactams bearing peptidoglycan-mimetic side chains have been determined. The structure of the hydrolysis product of an analogous peptide bound noncovalently to the former enzyme has also been obtained. The R39 DD-peptidase structures reveal the presence of a specific binding site for the D-alpha-aminopimelyl side chain, characteristic of the stem peptide of Actinomadura R39. This binding site features a hydrophobic cleft for the pimelyl methylene groups and strong hydrogen bonding to the polar terminus. Both of these active site elements are provided by amino acid side chains from two separate domains of the protein. In contrast, no clear electron density corresponding to the terminus of the peptidoglycan-mimetic side chains is present when these beta-lactams are covalently bound to PBP5. There is, therefore, no indication of a specific side-chain binding site in this enzyme. These results are in agreement with those from kinetics studies published earlier and support the general prediction made at the time of a direct correlation between kinetics and structural evidence. The essential high-molecular-mass PBPs have demonstrated, to date, no specific reactivity with peptidoglycan-mimetic peptide substrates and beta-lactam inhibitors and, thus, probably do not possess a specific substrate-binding site of the type demonstrated here with the R39 DD-peptidase. This striking deficiency may represent a sophisticated defense mechanism against low-molecular-mass substrate-analogue inhibitors/antibiotics; its discovery should focus new inhibitor design. [less ▲]

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See detail2-Aminopropane-1,2,3-tricarboxylic acid: Synthesis and co-crystallization with the class A beta-lactamase BS3 of Bacillus licheniformis.
Beck, Josephine; Sauvage, Eric ULg; Charlier, Paulette ULg et al

in Bioorganic & Medicinal Chemistry Letters (2008), 18(13), 3764-8

The title compound 4 has been prepared in four steps from ethylglycinate in 63% overall yield. This amino analog of citric acid has been co-crystallized with the class A beta-lactamase BS3 of Bacillus ... [more ▼]

The title compound 4 has been prepared in four steps from ethylglycinate in 63% overall yield. This amino analog of citric acid has been co-crystallized with the class A beta-lactamase BS3 of Bacillus licheniformis and the structure of the complex fully analyzed by X-ray diffraction. Tris-ethyl aminocitrate 3 and the free tris-acid 4 have been tested against a member beta-lactamase from all distinct subgroups. They are novel inhibitors of class A beta-lactamases, still modest but more potent than citrate and isocitrate. [less ▲]

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See detailCrystal structure of the Bacillus subtilis penicillin-binding protein 4a, and its complex with a peptidoglycan mimetic peptide
Sauvage, Eric ULg; Duez, Colette ULg; Herman, Raphaël ULg et al

in Journal of Molecular Biology (2007), 371(2), 528-539

The genome of Bacillus subtilis encodes 16 penicillin-binding proteins (PBPs) involved in the synthesis and/or remodelling of the peptidoglycan during the complex life cycle of this sporulating Gram ... [more ▼]

The genome of Bacillus subtilis encodes 16 penicillin-binding proteins (PBPs) involved in the synthesis and/or remodelling of the peptidoglycan during the complex life cycle of this sporulating Gram-positive rod-shaped bacterium. PBP4a (encoded by the dacC gene) is a low-molecular mass PBP clearly exhibiting in vitro DD-carboxypeptidase activity. We have solved the crystal structure of this protein alone and in complex with a peptide (D-alpha'-aminopymelyl-epsilon-D-alanyl-D-alanine) that mimics the C-terminal end of the Bacillus peptidoglycan stem peptide. PBP4a is composed of three domains: the penicillin-binding domain with a fold similar to the class A 13-lactamase structure and two domains inserted between the conserved motifs 1 and 2 characteristic of the penicillin-recognizing enzymes. The soaking of PBP4a in a solution Of D-alpha-aminopymelyl-epsilon-D-alanyl-D-alanine resulted in an adduct between PBP4a and a D-alpha-aminopimelyl-epsilon-D-alanine dipeptide and an unbound D-alanine, i.e. the products of acylation of PBP4a by D-alpha-aminopymelyl-epsilon-D-alanyl-D-alanine with the release of a D-alanine. The adduct also reveals a binding pocket specific to the diaminopimelic acid, the third residue of the peptidoglycan stem pentapeptide of B. subtilis. This pocket is specific for this class of PBPs. (C) 2007 Elsevier Ltd. All rights reserved. [less ▲]

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See detailReactivity of penicillin-binding proteins with peptidoglycan-mimetic beta-lactams: what's wrong with these enzymes?
Josephine, Helen R.; Charlier, Paulette ULg; Davies, Christopher et al

in Biochemistry (2006), 45(51), 15873-83

Beta-lactams exert their antibiotic action through their inhibition of bacterial DD-peptidases (penicillin-binding proteins). Bacteria, in general, carry several such enzymes localized on the outside of ... [more ▼]

Beta-lactams exert their antibiotic action through their inhibition of bacterial DD-peptidases (penicillin-binding proteins). Bacteria, in general, carry several such enzymes localized on the outside of their cell membrane to catalyze the final step in cell wall (peptidoglycan) synthesis. They have been classified into two major groups, one of high molecular weight, the other of low. Members of the former group act as transpeptidases in vivo, and their inhibition by beta-lactams leads to cessation of bacterial growth. The latter group consists of DD-carboxypeptidases, and their inhibition by beta-lactams is generally not fatal to bacteria. We have previously shown that representatives of the former group are ineffective at catalyzing the hydrolysis/aminolysis of peptidoglycan-mimetic peptides in vitro [Anderson et al. (2003) Biochem. J. 373, 949-955]. The theme of these experiments is expanded in the present paper where we describe the synthesis of a series of beta-lactams (penicillins and cephalosporins) containing peptidoglycan-mimetic side chains and the kinetics of their inhibition of a panel of penicillin-binding proteins spanning the major classes (Escherichia coli PBP 2 and PBP 5, Streptococcus pneumoniae PBP 1b, PBP 2x and PBP 3, the Actinomadura R39 DD-peptidase, and the Streptomyces R61 DD-peptidase). The results of these experiments mirror and expand the previous results with peptides. Neither peptides nor beta-lactams with appropriate peptidoglycan-mimetic side chains react with the solubilized constructs of membrane-bound penicillin binding proteins (the first five enzymes above) at rates exceeding those of generic analogues. Such peptides and beta-lactams do react at greatly enhanced rates with certain soluble low molecular weight enzymes (R61 and R39 DD-peptidases). The former result is unexpected and interesting. Why do the majority of penicillin-binding proteins not recognize elements of local peptidoglycan structure? Possible answers are discussed. That this question needs to be asked casts fascinating shadows on current studies of penicillin-binding proteins for new drug design. [less ▲]

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See detailCrystal structure of the Mycobacterium fortuitum class A beta-lactamase: structural basis for broad substrate specificity.
Sauvage, Eric ULg; Fonze, Eveline; Quinting, Birgit et al

in Antimicrobial Agents and Chemotherapy (2006), 50(7), 2516-21

beta-Lactamases are the main cause of bacterial resistance to penicillins and cephalosporins. Class A beta-lactamases, the largest group of beta-lactamases, have been found in many bacterial strains ... [more ▼]

beta-Lactamases are the main cause of bacterial resistance to penicillins and cephalosporins. Class A beta-lactamases, the largest group of beta-lactamases, have been found in many bacterial strains, including mycobacteria, for which no beta-lactamase structure has been previously reported. The crystal structure of the class A beta-lactamase from Mycobacterium fortuitum (MFO) has been solved at 2.13-A resolution. The enzyme is a chromosomally encoded broad-spectrum beta-lactamase with low specific activity on cefotaxime. Specific features of the active site of the class A beta-lactamase from M. fortuitum are consistent with its specificity profile. Arg278 and Ser237 favor cephalosporinase activity and could explain its broad substrate activity. The MFO active site presents similarities with the CTX-M type extended-spectrum beta-lactamases but lacks a specific feature of these enzymes, the VNYN motif (residues 103 to 106), which confers on CTX-M-type extended-spectrum beta-lactamases a more efficient cefotaximase activity. [less ▲]

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See detailCrystal structure of BRL 42715, C6-(N1-methyl-1,2,3-triazolylmethylene)penem, in complex with Enterobacter cloacae 908R beta-lactamase: evidence for a stereoselective mechanism from docking studies.
Michaux, Catherine; Charlier, Paulette ULg; Frère, Jean-Marie ULg et al

in Journal of the American Chemical Society (2005), 127(10), 3262-3

BRL 42715, C6-(N1-methyl-1,2,3-triazolylmethylene)penem, is an active-site-directed inactivator of bacterial beta-lactamases. The crystal structure of Enterobacter cloacae 908R class C beta-lactamase in ... [more ▼]

BRL 42715, C6-(N1-methyl-1,2,3-triazolylmethylene)penem, is an active-site-directed inactivator of bacterial beta-lactamases. The crystal structure of Enterobacter cloacae 908R class C beta-lactamase in complex with BRL 42715, docking, and energy minimization studies explain stereoselectivity of the binding of C6-(heterocyclic methylene)penems against class C beta-lactamase. [less ▲]

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See detailCrystal structure of the Actinomadura R39 DD-peptidase reveals new domains in penicillin-binding proteins.
Sauvage, Eric ULg; Herman, Raphaël ULg; Petrella, Stephanie et al

in Journal of Biological Chemistry (2005), 280(35), 31249-56

Actinomadura sp. R39 produces an exocellular DD-peptidase/penicillin-binding protein (PBP) whose primary structure is similar to that of Escherichia coli PBP4. It is characterized by a high beta-lactam ... [more ▼]

Actinomadura sp. R39 produces an exocellular DD-peptidase/penicillin-binding protein (PBP) whose primary structure is similar to that of Escherichia coli PBP4. It is characterized by a high beta-lactam-binding activity (second order rate constant for the acylation of the active site serine by benzylpenicillin: k2/K = 300 mm(-1) s(-1)). The crystal structure of the DD-peptidase from Actinomadura R39 was solved at a resolution of 1.8 angstroms by single anomalous dispersion at the cobalt resonance wavelength. The structure is composed of three domains: a penicillin-binding domain similar to the penicillin-binding domain of E. coli PBP5 and two domains of unknown function. In most multimodular PBPs, additional domains are generally located at the C or N termini of the penicillin-binding domain. In R39, the other two domains are inserted in the penicillin-binding domain, between the SXXK and SXN motifs, in a manner similar to "Matryoshka dolls." One of these domains is composed of a five-stranded beta-sheet with two helices on one side, and the other domain is a double three-stranded beta-sheet inserted in the previous domain. Additionally, the 2.4-angstroms structure of the acyl-enzyme complex of R39 with nitrocefin reveals the absence of active site conformational change upon binding the beta-lactams. [less ▲]

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See detailDesign, synthesis and evaluation of graftable thrombin inhibitors for the preparation of blood-compatible polymer materials.
Salvagnini, Claudio; Michaux, Catherine; Remiche, Julie ULg et al

in Organic & Biomolecular Chemistry (2005), 3(23), 4209-20

Piperazinyl-amide derivatives of N-alpha-(3-trifluoromethyl-benzenesulfonyl)-L-arginine (1) were synthesized as graftable thrombin inhibitors. The possible disturbance of biological activity due to a ... [more ▼]

Piperazinyl-amide derivatives of N-alpha-(3-trifluoromethyl-benzenesulfonyl)-L-arginine (1) were synthesized as graftable thrombin inhibitors. The possible disturbance of biological activity due to a variable spacer-arm fixed on the N-4 piperazinyl position was evaluated in vitro, against human alpha-thrombin, and in blood coagulation assay. Molecular modelling (in silico analysis) and X-ray diffraction studies of thrombin-inhibitor complexes were also performed. The fixation of bioactive molecules on poly(butylene terephthalate) (PBT) and poly(ethylene terephthalate) (PET) membranes was performed by wet chemistry treatment and evaluated by XPS analysis. Surface grafting of inhibitor 1d improved the membrane hemocompatibility by reducing blood clot formation on the modified surface. [less ▲]

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See detailCrystal structure of the sensor domain of the BlaR penicillin receptor from Bacillus licheniformis
Kerff, Frédéric ULg; Charlier, Paulette ULg; Colombo, Maria Louisa et al

in Biochemistry (2003), 42(44), 12835-12843

As in several staphylococci, the synthesis of the Bacillus licheniformis 749/I beta-lactamase is an inducible phenomenon regulated by a signal-transducing membrane protein BlaR. The C-terminal domain of ... [more ▼]

As in several staphylococci, the synthesis of the Bacillus licheniformis 749/I beta-lactamase is an inducible phenomenon regulated by a signal-transducing membrane protein BlaR. The C-terminal domain of this multimodular protein is an extracellular domain which specifically recognizes beta-lactam antibiotics. When it binds a beta-lactam, a signal is transmitted by the transmembrane region to the intracellular loops. In response, the hydrolytic activity of the BlaR large cytoplasmic L3 loop is induced, and a cascade of reactions is generated, leading to the transcription of the beta-lactamase gene. Here, we describe the crystal structure of the extracellular penicillin-receptor domain of BlaR (residues 346-601) at 2.5 Angstrom resolution in order to understand why this domain, whose folding is very similar to that of class D beta-lactamases, behaves as a highly sensitive penicillin-binding protein rather than a beta-lactamase. Two residues of the BlaR C-terminal domain, Thr452 and Thr542, modify the hydrophobic characteristic of the class D beta-lactamase active site. Both residues seem to be in part responsible for the lack of beta-lactamase activity of the BlaR protein due to the stability of the acyl-enzyme. Although further experimental data are needed to fully understand the transmembrane induction process, the comparison of the BlaR sensor domain structure with those of class D beta-lactamase complexes and penicillin-binding proteins provides interesting elements to hypothesize on possible signal transmission mechanisms. [less ▲]

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See detailOn the substrate specificity of bacterial DD-peptidases: evidence from two series of peptidoglycan-mimetic peptides
Anderson, J. W.; Adediran, S. A.; Charlier, Paulette ULg et al

in Biochemical Journal (2003), 373(Part 3), 949-955

The reactions between bacterial DD-peptidases and beta-lactam antibiotics have been studied for many years. Less well understood are the interactions between these enzymes and their natural substrates ... [more ▼]

The reactions between bacterial DD-peptidases and beta-lactam antibiotics have been studied for many years. Less well understood are the interactions between these enzymes and their natural substrates, presumably the peptide moieties of peptidoglycan. In general, remarkably little activity has previously been demonstrated in vitro against potential peptide substrates, although in many cases the peptides employed were non-specific and not homologous with the relevant peptidoglycan. In this paper, the specificity of a panel of DD-peptidases against elements of species-specific D-alanyl-D-alanine peptides has been assessed. In two cases, those of soluble, low-molecular-mass DD-peptidases, high activity against the relevant peptides has been demonstrated. In these cases, the high specificity is towards the free N-terminus of the peptidoglycan fragment. With a number of other enzymes, particularly high-molecular-mass DD-peptidases, little or no activity against these peptides was observed. In separate experiments, the reactivity of the enzymes against the central, largely invariant, peptide stem was examined. None of the enzymes surveyed showed high activity against this structural element although weak specificity in the expected direction towards the one structural variable (D-gammaGln versus D-gammaGlu) was observed. The current state of understanding of the activity of these enzymes in vitro is discussed. [less ▲]

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See detailCrystal structure of Enterobacter cloacae 908R class C beta-lactamase bound to iodo-acetamido-phenyl boronic acid, a transition-state analogue
Wouters, J.; Fonze, E.; Vermeire, M. et al

in Cellular and Molecular Life Sciences (2003), 60(8), 1764-1773

The structures of the, class C beta-lactamase from Enterobacter cloacae 908R alone and in complex with a baronic acid transition-state analogue were determined by X-ray crystallography at 2.1 and 2.3 ... [more ▼]

The structures of the, class C beta-lactamase from Enterobacter cloacae 908R alone and in complex with a baronic acid transition-state analogue were determined by X-ray crystallography at 2.1 and 2.3 Angstrom, respectively. The structure of the enzyme resembles those of other class C beta-lactamases. The structure of the. complex with the transition-state analogue, iodo-acetamido-phenyl boronic acid, shows that the inhibitor is covalently, bound to the active-site serine (Ser64). Binding of the inhibitor within the active site is compared with previously determined structures of complexes with other class C enzymes. The structure of the boronic acid adduct indicates ways to improve the affinity of this class of inhibitors. This structure of 908R class C beta-lactamase in complex with a transitionstate analogue provides further insights into the mechanism of action of these hydrolases. [less ▲]

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See detailCatalytic mechanism of the Streptomyces K15 DD-transpeptidase/penicillin-binding protein probed by site-directed mutagenesis and structural analysis
Rhazi, Noureddine ULg; Charlier, Paulette ULg; Dehareng, Dominique ULg et al

in Biochemistry (2003), 42(10), 2895-2906

The Streptomyces K15 penicillin-binding DD-transpeptidase is presumed to be involved in peptide cross-linking during bacterial cell wall peptidoglycan assembly. To gain insight into the catalytic ... [more ▼]

The Streptomyces K15 penicillin-binding DD-transpeptidase is presumed to be involved in peptide cross-linking during bacterial cell wall peptidoglycan assembly. To gain insight into the catalytic mechanism, the roles of residues Lys38, Ser96, and Cys98, belonging to the structural elements defining the active site cleft, have been investigated by site-directed mutagenesis, biochemical studies, and X-ray diffraction analysis. The Lys38His and Ser96Ala mutations almost completely abolished the penicillin binding and severely impaired the transpeptidase activities while the geometry of the active site was essentially the same as in the wild-type enzyme. It is proposed that Lys38 acts as the catalytic base that abstracts a proton from the active serine Ser35 during nucleophilic attack and that Ser96 is a key intermediate in the proton transfer from the Ogamma of Ser35 to the substrate leaving group nitrogen. The role of these two residues should be conserved among penicillin-binding proteins containing the Ser-Xaa-Asn/Cys sequence in motif 2. Conversion of Cys98 into Asn decreased the transpeptidase activity and increased hydrolysis of the thiolester substrate and the acylation rate with most beta-lactam antibiotics. Cys98 is proposed to play the same role as Asn in motif 2 of other penicilloyl serine transferases in properly positioning the substrate for the catalytic process. [less ▲]

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See detailCrystal structures of the Bacillus licheniformis BS3 class A beta-lactamase and of the acyl-enzyme adduct formed with cefoxitin
Fonzé, Evelyne; Vanhove, Mac; Dive, Georges ULg et al

in Biochemistry (2002), 41(6), 1877-1885

The Bacillus licheniformis BS3 beta-lactamase catalyzes the hydrolysis of the beta-lactam ring of penicillins, cephalosporins, and related compounds. The production of beta-lactamases is the most common ... [more ▼]

The Bacillus licheniformis BS3 beta-lactamase catalyzes the hydrolysis of the beta-lactam ring of penicillins, cephalosporins, and related compounds. The production of beta-lactamases is the most common and thoroughly studied cause of antibiotic resistance. Although they escape the hydrolytic activity of the prototypical Staphylococcus aureus beta-lactamase, many cephems are good substrates for a large number of beta-lactamases. However, the introduction of a 7alpha-methoxy substituent, as in cefoxitin, extends their antibacterial spectrum to many cephalosporin-resistant Gram-negative bacteria. The 7alphamethoxy group selectively reduces the hydrolytic action of many beta-lactamases without having a significant effect on the affinity for the target enzymes, the membrane penicillin-binding proteins. We report here the crystallographic structures of the BS3 enzyme and its acyl-enzyme adduct with cefoxitin at 1.7 Angstrom resolution. The comparison of the two structures reveals a covalent acyl-enzyme adduct with perturbed active site geometry, involving a different conformation of the Omega-loop that bears the essential catalytic Glu166 residue. This deformation is induced by the cefoxitin side chain whose position is constrained by the presence of the alpha-methoxy group. The hydrolytic water molecule is also removed from the active site by the 7beta-carbonyl of the acyl intermediate. In light of the interactions and steric hindrances in the active site of the structure of the BS3-cefoxitin acyl-enzyme adduct, the crucial role of the conserved Asn132 residue is confirmed and a better understanding of the kinetic results emerges. [less ▲]

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See detailThe 2.4-A crystal structure of the penicillin-resistant penicillin-binding protein PBP5fm from Enterococcus faecium in complex with benzylpenicillin.
Sauvage, Eric ULg; Kerff, Frédéric ULg; Fonze, E. et al

in Cellular and Molecular Life Sciences : CMLS (2002), 59(7), 1223-32

Penicillin-binding proteins (PBPs) are membrane proteins involved in the final stages of peptidoglycan synthesis and represent the targets of beta-lactam antibiotics. Enterococci are naturally resistant ... [more ▼]

Penicillin-binding proteins (PBPs) are membrane proteins involved in the final stages of peptidoglycan synthesis and represent the targets of beta-lactam antibiotics. Enterococci are naturally resistant to these antibiotics because they produce a PBP, named PBP5fm in Enterococcus faecium, with low-level affinity for beta-lactams. We report here the crystal structure of the acyl-enzyme complex of PBP5fm with benzylpenicillin at a resolution of 2.4 A. A characteristic of the active site, which distinguishes PBP5fm from other PBPs of known structure, is the topology of the loop 451-465 defining the left edge of the cavity. The residue Arg464, involved in a salt bridge with the residue Asp481, confers a greater rigidity to the PBP5fm active site. In addition, the presence of the Val465 residue, which points into the active site, reducing its accessibility, could account for the low affinity of PBP5fm for beta-lactam. This loop is common to PBPs of low affinity, such as PBP2a from Staphylococcus aureus and PBP3 from Bacillus subtilis. Moreover, the insertion of a serine after residue 466 in the most resistant strains underlines even more the determining role of this loop in the recognition of the substrates. [less ▲]

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