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Serine beta-lactamases contribute widely to the beta-lactam resistance phenomena. Unfortunately, the intimate details of their catalytic mechanism remain elusive and subject to some controversy even though many "natural" and "artificial" mutants of these different enzymes have been isolated. This paper is essentially focused on class C beta-lactamases, which contain a Tyr (Tyr150) as the first residue of the second conserved element, in contrast to their class A counterparts, in which a Ser is found in the corresponding position. We have modified this Tyr residue by site-directed mutagenesis. On the basis of the three-dimensional structure of the Enterobacter cloacae P99 enzyme, it seemed that residues Glu272 and His314 might also be important. They were similarly substituted. The modified enzymes were isolated and their catalytic properties determined. Our results indicated that His314 was not required for catalysis and that Glu272 did not play an important role in acylation but was involved to a small extent in the deacylation process. Conversely, Tyr150 was confirmed to be central for catalysis, at least with the best substrates. On the basis of a comparison of data obtained for several class C enzyme mutants and in agreement with recent structural data, we propose that the phenolate anion of Tyr150, in conjunction with the alkyl ammonium of Lys315, acts as the general base responsible for the activation of the active-site Ser64 during the acylation step and for the subsequent activation of a water molecule in the deacylation process. The evolution of the important superfamily of penicillin-recognizing enzymes is further discussed in the light of this proposed mechanism.

The sensitivity of a bacterium to β-lactam antibiotics depends upon the interplay between 3 independent factors: the sensitivity of the essential penicillin-binding enzyme(s), thee quantity and properties of the β-lactamase(s) and the diffusion barrier that the outer-membrane of Gram-negative bacteria can represent. Those three factors can be modified by mutations or by the horizontal transfer of genes or portions of genes.

Bacillus subtilis DppA is a binuclear zinc-dependent, D-specific aminopeptidase. The X-ray structure of the enzyme has been determined at 2.4 A resolution by a three-wavelength MAD experiment. The structure reveals that DppA is a new example of a 'self-compartmentalizing protease', a family of proteolytic complexes. Proteasomes are the most extensively studied representatives of this family. The DppA enzyme is composed of identical 30 kDa subunits organized in a decamer with 52 point-group symmetry. A 20 A wide channel runs through the complex, giving access to a central chamber holding the active sites. The structure shows DppA to be a prototype of a new family of metalloaminopeptidases characterized by the SXDXEG key sequence.

Incubation of moxalactam and cefoxitin with the Aeromonas hydrophila metallo-beta-lactamase CphA leads to enzyme-catalyzed hydrolysis of both compounds and to irreversible inactivation of the enzyme by the reaction products. As shown by electrospray mass spectrometry, the inactivation of CphA by cefoxitin and moxalactam is accompanied by the formation of stable adducts with mass increases of 445 and 111 Da, respectively. The single thiol group of the inactivated enzyme is no longer titrable, and dithiothreitol treatment of the complexes partially restores the catalytic activity. The mechanism of inactivation by moxalactam was studied in detail. Hydrolysis of moxalactam is followed by elimination of the 3' leaving group (5-mercapto-1-methyltetrazole), which forms a disulfide bond with the cysteine residue of CphA located in the active site. Interestingly, this reaction is catalyzed by cacodylate.

The DmpA (d-aminopeptidase A) protein produced by Ochrobactrum anthropi hydrolyses p-nitroanilide derivatives of glycine and d-alanine more efficiently than that of l-alanine. When regular peptides are utilized as substrates, the enzyme behaves as an aminopeptidase with a preference for N-terminal residues in an l configuration, thus exemplifying an interesting case of stereospecificity reversal. The best-hydrolysed substrate is l-Ala-Gly-Gly, but tetra- and penta-peptides are also efficiently hydrolysed. The gene encodes a 375-residue precursor, but the active enzyme contains two polypeptides corresponding to residues 2-249 (alpha-subunit) and 250-375 (beta-subunit) of the precursor. Residues 249 and 250 are a Gly and a Ser respectively, and various substitutions performed by site-directed mutagenesis result in the production of an uncleaved and inactive protein. The N-terminal Ser residue of the beta-subunit is followed by a hydrophobic peptide, which is predicted to form a beta-strand structure. All these properties strongly suggest that DmpA is an N-terminal amidohydrolase. An exploration of the databases highlights the presence of a number of open reading frames encoding related proteins in various bacterial genomes. Thus DmpA is very probably the prototype of an original family of N-terminal hydrolases.

In addition to the BlaB metallo-beta-lactamase, Chryseobacterium (Flavobacterium) meningosepticum CCUG 4310 (NCTC 10585) constitutively produces a 31-kDa active-site serine beta-lactamase, named CME-1, with an alkaline isoelectric pH. The blaA(CME) gene that encodes the latter enzyme was isolated from a genomic library constructed in the Escherichia coli plasmid vector pACYC184 by screening for cefuroxime-resistant clones. Sequence analysis revealed that the CME-1 enzyme is a new class A beta-lactamase structurally divergent from the other members of this class, being most closely related to the VEB-1 (also named CEF-1) and PER beta-lactamases and the Bacteroides chromosomal cephalosporinases. The blaA(CME) determinant is located on the chromosome and exhibits features typical of those of C. meningosepticum resident genes. The CME-1 protein was purified from an E. coli strain that overexpresses the cloned gene via a T7-based expression system by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. Kinetic parameters for several substrates were determined. CME-1 is a clavulanic acid-susceptible extended-spectrum beta-lactamase that hydrolyzes most cephalosporins, penicillins, and monobactams but that does not hydrolyze cephamycins and carbapenems. The enzyme exhibits strikingly different kinetic parameters for different classes of beta-lactams, with both K(m) and k(cat) values much higher for cephalosporins than for penicillins and monobactams. However, the variability of both kinetic parameters resulted in overall similar acylation rates (k(cat)/K(m) ratios) for all types of beta-lactam substrates.

The investigation of a proposed glueball filter in proton-proton collisions led us to show that its action is in fact easily understood in terms of kinematics. While the procedure proposed stays of interest in reducing background when lookingfor centrally produced resonances of specified spin and parity, we stress that it can be put to advantage in giving access to the polarization of gluons in the inital protons, Comment: talk presented at QCD98, Montepellier. to appear in proceedings

Motivated by a pilot experiment conducted by F.Vannucci et al. during a solar eclipse, we work out the geometry governing the radiative decays of solar neutrinos. Surprisingly, although a smaller proportion of the photons can be detected, the case of strongly non-degenerate neutrinos brings better limits in terms of the fundamental couplings. We advocate satellite-based experiments to improve the sensitivity., Comment: 11 pages, 2 Postscript figures

The catalytic pathway of class A beta-lactamases involves an acyl-enzyme intermediate where the substrate is ester-linked to the Ser-70 residue. Glu-166 and Lys-73 have been proposed as candidates for the role of general base in the activation of the serine OH group. The replacement of Glu-166 by an asparagine in the TEM-1 and by a histidine in the Streptomyces albus G beta-lactamases yielded enzymes forming stable acyl-enzymes with beta-lactam antibiotics. Although acylation of the modified proteins by benzylpenicillin remained relatively fast, it was significantly impaired when compared to that observed with the wild-type enzyme. Moreover, the E166N substitution resulted in a spectacular modification of the substrate profile much larger than that described for other mutations of Omega-loop residues. Molecular modeling studies indicate that the displacement of the catalytic water molecule can be related to this observation. These results confirm the crucial roles of Glu-166 and of the "catalytic" water molecule in both the acylation and the deacylation processes.

Bacterial resistance to beta-lactam antibiotics, a clinically worrying and recurrent problem, is often due to the production of beta-lactamases, enzymes that efficiently hydrolyze the amide bond of the beta-lactam nucleus. Imipenem and other carbapenems escape the activity of most active site serine beta-lactamases and have therefore become very popular drugs for antibacterial chemotherapy in the hospital environment. Their usefulness is, however, threatened by the appearance of new beta-lactamases that efficiently hydrolyze them. This study is focused on the structure and properties of two recently described class A carbapenemases, produced by Serratia marcescens and Enterobacter cloacae strains and leads to a better understanding of the specificity of beta-lactamases. In turn, this will contribute to the design of better antibacterial drugs. Three-dimensional models of the two class A carbapenemases were constructed by homology modeling. They suggested the presence, near the active site of the enzymes, of a disulfide bridge (C69-C238) whose existence was experimentally confirmed. Kinetic parameters were measured with the purified Sme-1 carbapenemase, and an attempt was made to explain its specific substrate profile by analyzing the structures of minimized Henri-Michaelis complexes and comparing them to those obtained for the "classical" TEM-1 beta-lactamase. The peculiar substrate profile of the carbapenemases appears to be strongly correlated with the presence of the disulfide bridge between C69 and C238.

The stability and kinetics of unfolding and refolding of the P167T mutant of the TEM-1 beta-lactamase have been investigated as a function of guanidine hydrochloride concentration. The activity of the mutant enzyme was not significantly modified, which strongly suggests that the Glu166-Thr167 peptide bond, like the Glu166-Pro167, is cis. The mutation, however, led to a significant decrease in the stability of the native state relative to both the thermodynamically stable intermediate and the fully unfolded state of the protein. In contrast to the two slower phases seen in the refolding of the wild-type enzyme, only one phase was detected in the refolding of the mutant, indicating a determining role of proline 167 in the kinetics of folding of the wild-type enzyme. The former phases are replaced by rapid refolding when the enzyme is unfolded for short periods of time, but the latter is independent of the time of unfolding. The monophasic refolding reaction of the mutant is proposed to reflect mainly the trans--cis isomerization of the Glu166-Thr167 peptide bond.

The catalytic properties of six "natural" mutants of the TEM-1 beta-lactamase have been studied in detail, with special emphasis on their activity versus third-generation cephalosporins. On the basis of the recently determined high-resolution structure of the wild-type enzyme, and of the substrates' structures optimized by the AMI quantum chemistry method, we have attempted to explain the influences of the mutations on the substrate profiles of the enzymes. Some of the kinetic results have thus received a satisfactory, semi-quantitative interpretation, especially in the case of single mutations. Analysis of the double mutants proved more hazardous. Extending the comparison to some other class A beta-lactamases showed that similar properties could result from different sequences, supplying an interesting example of convergent evolution within a generally diverging family.

A cysteine residue has been substituted for the active-site serine of the class-C beta-lactamase produced by Enterobacter cloacae 908R by site-directed mutagenesis. The modified protein exhibited drastically reduced kcat./Km values on all tested substrates. However, this decrease was due to increased Km values with some substrates and to decreased kcat. values with others. These apparently contradictory results could be explained by a selective influence of the mutation on the first-order rate constant characteristic of the acylation step, a hypothesis which was confirmed by the absence of detectable acylenzyme accumulation with all the tested substrates, with the sole exception of cefoxitin.

The involvement of the serine residue 318 in the specificity of a class C beta-lactamase was investigated. Multiple site-directed mutants at this position were generated using a polymerase chain reaction technique. These mutants were then probed for their activity towards various beta-lactam compounds. One mutant, S318G was further purified and its physico-chemical and catalytic properties determined. It was shown that the observed minimal inhibitory concentration values of this mutant could be correlated to its kinetic properties using a 'diffusion-hydrolysis' model. However, the data showed that residue 318 has little influence on the specificity of class C beta-lactamases.

In an effective Lagrangian approach to physics beyond the Standard Model, it has been argued that imposing $SU(2) \times U(1)$ invariance severely restricts the discovery potential of future colliders. We exhibit a possible way out in an extended gauge group context., Comment: 14 pages , CERN-TH.6573/92 ULB.TH.04/92 (phyzzx, 3 eps-figs incl.)

A non-steady-state model is discussed for the study of the interplay between beta-lactamase activity and outer membrane permeability with slowly hydrolysed beta-lactams. The analysis shows: (1) that the simple, steady-state model presented in the accompanying paper remains valid as long as kcat (i.e. k3 with chromosome-encoded class C beta-lactamases) is larger than 10(-3)/sec (generation time = 20 min or more); (2) that among the beta-lactam antibiotics studied here, the complete, non-steady-state model needs only be used in the case of aztreonam; (3) that the term "trapping" should be replaced by "formation of a covalent acyl-enzyme" and that such a phenomenon only contributes significantly to the resistance when penetration and hydrolysis are very slow and the periplasmic beta-lactamase concentration is very high. Aztreonam seems to be the only compound which fulfils the first two conditions.

The interplay between the three factors involved in the resistance of bacteria to beta-lactam antibiotics (sensitivity of target, synthesis of beta-lactamase, permeability barrier) is analysed and discussed on the basis of a simple kinetic model. The three factors do not act independently. In Gram-negative bacteria, the permeability barrier is only significant when the bacterial cell also produces a beta-lactamase. Special attention is devoted to cases where large periplasmic beta-lactamase concentrations prevail, a situation which has been observed in some clinical isolates.

The beta-lactamases (EC 3.5.2.6) from TEM plasmid RP4, Bacillus licheniformis 749/C and Enterobacter cloacae P99 were studied in solution over a wide concentration range by equilibrium sedimentation. Though crystal symmetries indicate that all three enzymes are potentially dimeric in their crystal forms, in 50 mM-sodium cacodylate at pH 6.5 the enzymes show only a small tendency to associate, indicated by a weight-average Mr (Mw) at 3% (w/v) concentration about 9% greater than that of the monomer. Although the mode of association could not be determined, this extent of association corresponded to a dimerization constant of about 2 × 10(2) M-1. In 2.1 M-(NH4)2SO4 the B. licheniformis enzyme shows some association at concentrations over 1%, displaying an Mw value at 7% concentration about 60% more than the monomer. Under the same conditions Mw for the Entero. P99 enzyme is about 60% greater than the monomer near the solubility limit of about 2%. However, the Mw for the TEM enzyme is over twice that of the monomer at its solubility limit (3%) in 1.7 M-(NH4)2SO4. Fitting the sedimentation data of the TEM enzyme in 1.7 M-(NH4)2SO4 with a dimerization model and an indefinite-isodesmic-association model yielded equilibrium constants of 1.5 × 10(4) and 3.3 × 10(2) M-1 respectively, with the indefinite-isodesmic model giving the better fit. Fitting the data for the other two enzymes yielded values of 1.4 × 10(3) and 1.7 × 10(2) M-1 respectively for the Entero. P99 enzyme and 4.5 × 10(2) and 45 M-1 respectively for the B. licheniformis enzyme. It could not be determined which model was the better fit for these two enzymes. Since none of the beta-lactamases studied here showed strong evidence of the terminal aggregate being a dimer, we conclude that crystalline dimers, if they exist, will not be tightly associated or physiologically significant.

The gene encoding the extracellular, active-site serine beta-lactamase of Actinomadura R39, previously cloned into Streptomyces lividans, has the information for the synthesis of a 304 amino acid protein, the amino terminal region of which has the characteristic features of a signal peptide. The Actinomadura R39 beta-lactamase is another member of the class A beta-lactamases. In particular, it shows high homology with the beta-lactamase of Bacillus licheniformis.

The beta-lactamase from Enterobacter cloacae P99 has been crystallized from polyethylene glycol solution at pH 7. X-ray examination of the orthorhombic crystals shows the space group is P2(1)2(1)2 with unit cell dimensions a = 77.4 A, b = 69.4 A, and c = 63.6 A. There is one molecule of molecular weight 39,000 in the asymmetric unit.

In the presence of a reporter substrate, the progressive inactivation of an enzyme was easily studied by directly transmitting absorbance readings to a microcomputer. Pseudo-first order rate constants as high as 0.3 sec-1 were rapidly and accurately measured. When utilization of the reporter substrate did not exceed 10%, the rate of the reaction (vt) could be considered as proportional to the active enzyme concentration at any time during the analysis and the decrease of vt was first order with time. This simple method was used to follow the inactivation of beta-lactamases (EC 3.5.2.6) by various physical and chemical agents. When a large proportion (30-80%) of reporter substrate was destroyed, a correction was introduced to account for the corresponding decrease of its rate of utilization. This enabled experiments to be performed with a DD-peptidase and a substrate exhibiting a low delta epsilon upon hydrolysis. For the first time, the inactivation of a penicillin-sensitive enzyme by a beta-lactam could be continuously and directly observed. Finally, the method was extended to the study of hysteresis phenomena.

Wall peptidoglycan expansion in bacteria rests upon a cytoplasmic D-Ala: D-Ala ligase (ADP) which catalyses synthesis of a D-Ala-D-Ala dipeptide (with accompanying hydrolysis of one molecule of ATP) and a set of DD-peptidases which utilize this D-Ala-D-Ala dipeptide--once it has been translocated at the outer face of the plasma membrane as the C-terminal portion of a disaccharide peptide unit--as carbonyl donor for transpeptidation and carboxypeptidation reactions (without additional energy expenditure). Four DD-peptidases have been selected which differ from each other with respect to the effects that amino compounds exert on the fate and rate of consumption of a D-Ala-D-Ala terminated amide carbonyl donor analogue. They serve as models to understand the different mechanisms by which the DD-peptidases perform catalysis and show widely varying responses to the action of beta-lactams, from extreme sensitivity to very high resistance.

The crystallographic structure of the penicillin-sensitive D-alanyl carboxypeptidase-transpeptidase from Streptomyces R61 has been solved to 2.8-A resolution. The 38,000-dalton serine peptidase has two regions of secondary structure, an alpha/beta cluster, and a region which contains five helical segments. The beta sheet is composed of five beta strands. The tertiary structure has no homology with the classic serine proteases or with the zinc carboxypeptidases. The binding at a common site of three types of beta-lactam (a penicillin, a cephalosporin, a monocyclic beta-lactam) and a desazacyclobutanone has been observed in Fourier difference maps. The binding site sequence is Val-Gly-Ser-Val-Thr-Lys. The beta-lactam ring lies near the enzyme's catalytic serine at position 37, and the C3 substituent of a cephalosporin falls near lysine 40.

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International audience; The commissioning of the ATLAS calorimeters is an ongoing process since early 2006. During this period, cosmic muons have been recorded in several runs combining both hadronic and electromagnetic calorimeters. Among the goals are the measuremement of the uniformity of the liquid argon electromagnetic calorimeter to the level of 1% and the intercalibration in time of its channels to 1 ns.

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Results from the CDF and D0 collaborations from searches of physics beyond the standard model are presented in reactions involving high transverse momentum photons or jets in their final states., Comment: Latex, 6 pages, 11 eps figures

International audience; The BiPo pro ject is dedicated to the measurement of extremely low radioactive contaminations of SuperNEMO ββ source foils ( 208 Tl < 2 µBq/kg and 214 Bi < 10 µBq/kg). A modular BiPo1 prototype with its 20 modules and its shielding test facility is running in the Modane Underground Laboratory since February, 2008. The goal of this prototype is to study the back-grounds and particularly the surface contamination of plastic scintillators. After 2 months, a preliminary upper limit on the sensitivity of a 10 m 2 BiPo detector in 208 Tl contamination of selenium source foils can be extrapolated to: A( 208 Tl) < 7.5 µBq/kg (90 % C.L.).

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International audience; γ /φ3 is the less precisely known of the Unitarity Triangle angles. The general problematics of measurements of this parameter are discussed and recent experimental results from Babar and Belle are presented.

on behalf of ATLAS and CMS collaborations; International audience; With only a few months until the LHC start-up, the commissioning of ATLAS is in its final stage as the last components of the detector are installed. The understanding of the detector response acquired during the preparation phase is presented as well as the expected performance at start-up. The strategies of both ATLAS and CMS regarding the use of early data involving leptons is then described. Assuming an integrated luminosity of 100 pb−1 in 2008, examples of calibration procedures and early measurements are given.

Within the framework of Standard Model, the production mode of Higgs boson through the fusion of the vector bosons $W$ or $Z$ (Vector Boson Fusion) is one of the most important production mechanisms, providing a specific detection signature. A detailed study regarding this issue is being undergone for ATLAS detector in LHC and some general features of this analysis are being presented in this note emphasizing in the study of Central Jet Veto., Comment: Proceedings for Moriond ElectroWeak 2008

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International audience; The current status of the experimental measurements and theoretical predictions of the anomalous magnetic moment of the muon $a\mu$ is briefly reviewed. The emphasis is put on the evaluation of the hadronic contribution to $a\mu$ as it has the largest uncertainty among all Standard Model contributions. The precision of the hadronic contribution is driven by the input $e^+e- $ data predominantly from the $\pi^+\pi^-$ channel. Including the latest experimental data on $e^+e^-$ annihilation into hadrons from CMD2 and SND for the $\pi^+\pi^-$ channel and BaBar for multi-hadron final states, the updated Standard Model prediction disagrees with the measurement dominated by BNL by 3.3 standard deviations, with the theoretical precision exceeding the experimental one.