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Biomedical subjects

P Charlier

Publications and source records attributed to P Charlier.

At least 19 recordsLinked to original sources
PubMed2003

Crystal structure of Enterobacter cloacae 908R class C beta-lactamase bound to iodo-acetamido-phenyl boronic acid, a transition-state analogue.

The structures of the class C beta-lactamase from Enterobacter cloacae 908R alone and in complex with a boronic acid transition-state analogue were determined by X-ray crystallography at 2.1 and 2.3 A, 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 transition-state analogue provides further insights into the mechanism of action of these hydrolases.

Apoenzymes↗
PubMed2002

The 2.4-A crystal structure of the penicillin-resistant penicillin-binding protein PBP5fm from Enterococcus faecium in complex with benzylpenicillin.

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.

Amino Acid Sequence↗
PubMed2002

[Chloroquine cardiomyopathy revealed by complete atrio-ventricular block. A case report].

We describe a rare case of chloroquine cardiomyopathy occurring during long term (7 years) treatment for rheumatoid polyarthritis in a 42 year old woman. There was an isolated acute severe conduction defect, which is particularly rare. Histological study with the electron microscope allowed confirmation of this diagnosis. We report here the secondary cardiological effects of this frequently used synthetic antimalarial.

Acute Disease↗
PubMed2001

Crystallographic analysis of family 11 endo-beta-1,4-xylanase Xyl1 from Streptomyces sp. S38.

Family 11 endo-beta-1,4-xylanases degrade xylan, the main constituent of plant hemicelluloses, and have many potential uses in biotechnology. The structure of Xyl1, a family 11 endo-xylanase from Streptomyces sp. S38, has been solved. The protein crystallized from ammonium sulfate in the trigonal space group P321, with unit-cell parameters a = b = 71.49, c = 130.30 A, gamma = 120.0 degrees. The structure was solved at 2.0 A by X-ray crystallography using the molecular-replacement method and refined to a final R factor of 18.5% (R(free) = 26.9%). Xyl1 has the overall fold characteristic of family 11 xylanases, with two highly twisted beta-sheets defining a long cleft containing the two catalytic residues Glu87 and Glu177.

Amino Acid Sequence↗
PubMed2001

Expression, purification, crystallization and preliminary X-ray analysis of the native class C beta-lactamase from Enterobacter cloacae 908R and two mutants.

Crystals have been obtained of the Enterobacter cloacae 908R beta-lactamase and two point mutants by the vapour-diffusion method using similar conditions [pH 9.0, polyethylene glycol (M(r) = 6000) as precipitant]. The three crystal forms belong to the orthorhombic space group P2(1)2(1)2, with roughly the same unit-cell parameters; i.e. for the wild-type crystals a = 46.46, b = 82.96, c = 95.31 A. In the best cases, the crystals diffract to about 2.1 A resolution on a rotating-anode X-ray source at room temperature. Co-crystallization experiments of poor substrates with the wild-type protein and the active-site serine mutant (S64C) are planned and should lead to a better understanding of the catalytic mechanism of class C beta-lactamases.

Crystallization↗
PubMed1999

The crystal structure of a penicilloyl-serine transferase of intermediate penicillin sensitivity. The DD-transpeptidase of streptomyces K15.

The serine DD-transpeptidase/penicillin-binding protein of Streptomyces K15 catalyzes peptide bond formation in a way that mimics the penicillin-sensitive peptide cross-linking reaction involved in bacterial cell wall peptidoglycan assembly. The Streptomyces K15 enzyme is peculiar in that it can be considered as an intermediate between classical penicillin-binding proteins, for which benzylpenicillin is a very efficient inactivator, and the resistant penicillin-binding proteins that have a low penicillin affinity. With its moderate penicillin sensitivity, the Streptomyces K15 DD-transpeptidase would be helpful in the understanding of the structure-activity relationship of this penicillin-recognizing protein superfamily. The structure of the Streptomyces K15 enzyme has been determined by x-ray crystallography at 2.0-A resolution and refined to an R-factor of 18.6%. The fold adopted by this 262-amino acid polypeptide generates a two-domain structure that is close to those of class A beta-lactamases. However, the Streptomyces K15 enzyme has two particular structural features. It lacks the amino-terminal alpha-helix found in the other penicilloyl-serine transferases, and it exhibits, at its surface, an additional four-stranded beta-sheet. These two characteristics might serve to anchor the enzyme in the plasma membrane. The overall topology of the catalytic pocket of the Streptomyces K15 enzyme is also comparable to that of the class A beta-lactamases, except that the Omega-loop, which bears the essential catalytic Glu(166) residue in the class A beta-lactamases, is entirely modified. This loop adopts a conformation similar to those found in the Streptomyces R61 DD-carboxypeptidase and class C beta-lactamases, with no equivalent acidic residue.

Amino Acid Sequence↗
PubMed1998

X-ray studies of enzymes that interact with penicillins.

The technique of X-ray diffraction has been successfully applied to enzymes associated with peptidoglycan biosynthesis. The technique has taught us a great deal about the structures and catalytic mechanisms of penicillin-binding proteins and beta-lactamases. An insight into the structural basis for antibiotic resistance is given.

Bacterial Proteins↗
PubMed1998

[A case of Enterobius vermicularis salpingitis].

Enterobius vermicularis peritonitis due to intestinal infection is a common entity described in the literature. We report here an unusual case of unilateral salpingitis associated with the parasite migration through the genital tract.

Adult↗
PubMed1997

Unexpected influence of a C-terminal-fused His-tag on the processing of an enzyme and on the kinetic and folding parameters.

The addition of a poly-His C-terminal extension, designed to facilitate the purification of the protein, to the beta-lactamase of a thermophilic Bacillus licheniformis strain modified the site of action of the signal peptidase. This resulted in the secretion of a protein with a different N-terminus, showing that this type of protein engineering might not always be as 'neutral' as generally assumed.

Bacillus↗
PubMed1996

The catalytic mechanism of beta-lactamases: NMR titration of an active-site lysine residue of the TEM-1 enzyme.

Beta-Lactamases are widespread in the bacterial world, where they are responsible for resistance to penicillins, cephalosporins, and related compounds, currently the most widely used antibacterial agents. Detailed structural and mechanistic understanding of these enzymes can be expected to guide the design of new antibacterial compounds resistant to their action. A number of high-resolution structures are available for class A beta-lactamases, whose catalytic mechanism involves the acylation of a serine residue at the active site. The identity of the general base which participates in the activation of this serine residue during catalysis has been the subject of controversy, both a lysine residue and a glutamic acid residue having been proposed as candidates for this role. We have used the pH dependence of chemical modification of epsilon-amino groups by 2,4,6,-trinitrobenzenesulfonate and the pH dependence of the epsilon-methylene 1H and 13C chemical shifts (in enzyme selectively labeled with [epsilon-13C]lysine) to estimate the pKa of the relevant lysine residue, lysine-73, of TEM-1 beta-lactamase. Both methods show that the pKa of this residue is > 10, making it very unlikely that this residue could act as a proton acceptor in catalysis. An alternative mechanism in which this role is performed by glutamate-166 through an intervening water molecule is described.

Binding Sites↗
PubMed1996

Penicillin and beyond: evolution, protein fold, multimodular polypeptides, and multiprotein complexes.

As the protein sequence and structure databases expand, the relationships between proteins, the notion of protein superfamily, and the driving forces of evolution are better understood. Key steps of the synthesis of the bacterial cell wall peptidoglycan are revisited in light of these advances. The reactions through which the D-alanyl-D-alanine depeptide is formed, utilized, and hydrolyzed and the sites of action of the glycopeptide and beta-lactam antibiotics illustrate the concept according to which new enzyme functions evolve as a result of tinkering of existing proteins. This occurs by the acquisition of local structural changes, the fusion into multimodular polypeptides, and the association into multiprotein complexes.

Bacterial Proteins↗
PubMed1995

TEM1 beta-lactamase structure solved by molecular replacement and refined structure of the S235A mutant.

beta-Lactamases are bacterial enzymes which catalyse the hydrolysis of the beta-lactam ring of penicillins, cephalosporins and related compounds, thus inactivating these antibiotics. The crystal structure of the TEM1 beta-lactamase has been determined at 1.9 A resolution by the molecular-replacement method, using the atomic coordinates of two homologous beta-lactamase refined structures which show about 36% strict identity in their amino-acid sequences and 1.96 A r.m.s. deviation between equivalent Calpha atoms. The TEM1 enzyme crystallizes in space group P2(1)2(1)2(1) and there is one molecule per asymmetric unit. The structure was refined by simulated annealing to an R-factor of 15.6% for 15 086 reflections with I >/= 2sigma(I) in the resolution range 5.0-1.9 A. The final crystallographic structure contains 263 amino-acid residues, one sulfate anion in the catalytic cleft and 135 water molecules per asymmetric unit. The folding is very similar to that of the other known class A beta-lactamases. It consists of two domains, the first is formed by a five-stranded beta-sheet covered by three alpha-helices on one face and one alpha-helix on the other, the second domain contains mainly alpha-helices. The catalytic cleft is located at the interface between the two domains. We also report the crystallographic study of the TEM S235A mutant. This mutation of an active-site residue specifically decreases the acylation rate of cephalosporins. This TEM S235A mutant crystallizes under the same conditions as the wild-type protein and its structure was refined at 2.0 A resolution with an R value of 17.6%. The major modification is the appearance of a water molecule near the mutated residue, which is incompatible with the OG 235 present in the wild-type enzyme, and causes very small perturbations in the interaction network in the active site.

Journal Article↗
PubMed1994

Crystallization and X-ray diffraction study of the Streptomyces K15 penicillin-binding DD-transpeptidase.

The 262 amino acid residue long DD-transpeptidase/penicillin-binding protein of Streptomyces K15 has been crystallized at room temperature by using the hanging drop vapour diffusion technique. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters a = 46.4 A, b = 54.1 A and c = 108.3 A. They contain one protein molecule per asymmetric unit and diffract to about 1.9 A. X-ray data have been collected to 2.0 A from a native crystal. The previously published amino acid sequence of the protein has been corrected at positions 71, 72, 113, 114 and 156.

Bacterial Proteins↗
PubMed1994

Direct n.m.r. evidence for substrate-induced conformational changes in a beta-lactamase.

Cefoxitin and other beta-lactam antibiotics with a methoxy group on the alpha-face behave as very poor substrates of the Bacillus licheniformis beta-lactamase. The kinetic properties of the enzyme-cefoxitin system made it theoretically suitable for a detailed structural study of the acyl-enzyme. Unfortunately, soaking the crystals in cefoxitin solution did not allow detection of a crystalline acyl-enzyme complex. In contrast, direct observation by n.m.r. of the stable acyl-enzyme formed with cefoxitin and moxalactam indicated clear modifications of the enzyme structure, which were reflected in the aromatic and high-field methyl regions of the spectrum. The return to the initial free enzyme spectrum was concomitant with the hydrolysis of the acyl-enzyme, the process being slow enough to allow multidimensional n.m.r. experiments.

Anti-Bacterial Agents↗
PubMed1993

Crystallization of a genetically engineered water-soluble primary penicillin target enzyme. The high molecular mass PBP2x of Streptococcus pneumoniae.

A genetically engineered water-soluble derivative of PBP2x of Streptococcus pneumoniae has been produced, purified and crystallized in a form suitable for X-ray diffraction analysis. The best crystals have been grown at 15 degrees C, from solutions containing 8% polyethylene glycol 10,000 at pH values ranging from 3.9 to 6.0. These crystals diffract to a resolution of 3.5 A and have a space group P6(1)22 (or enantiomorph) with unit cell dimensions of a = b = 162.2 A, c = 171.8 A, alpha = beta = 90 degrees, gamma = 120 degrees. The molecular mass and cell dimensions suggest that there is one molecule of enzyme per asymmetric unit. The breakdown of a chromogenic cephalosporin derivative diffused into a crystal reveals clearly that the enzyme is active in the crystalline state.

Bacterial Proteins↗
PubMed1992

Evaluation of an atrophic rhinitis vaccine under controlled conditions.

A vaccine containing inactivated cultures of Bordetella bronchiseptica, toxigenic Pasteurella multocida type D and dermonecrotic P multocida type D toxoid in an oil-in-water adjuvant was given to seven sows, with seven others acting as controls. Half the piglets in each litter were exposed intranasally when four days old to B bronchiseptica and when eight days old to toxigenic P multocida type D. There was considerably less sneezing in the litters of the vaccinated sows and when the piglets were 10 weeks old, only 18 per cent had deformed snouts compared with 74 per cent in the litters of the control sows. The average liveweight gain of the piglets born to vaccinated sows was significantly better (P less than 0.05) between two and 10 weeks of age than that of the piglets born to unvaccinated sows, although there were no significant lower respiratory tract lesions in either group. The conchal atrophy scores were significantly lower (P less than 0.001) in the piglets from the vaccinated sows and were negatively correlated (r = -0.37) with increasing liveweight gain. In the liters of the vaccinated sows, P multocida was not isolated from the nasal passages of the in-contact piglets and from only 7 per cent of those deliberately exposed compared with 65 per cent and 79 per cent, respectively, in the litters of the control sows. P multocida was isolated post mortem from the tonsils of 23 per cent of the piglets of vaccinated sows and from 87 per cent of those from unvaccinated sows.

Animals↗