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See detailSynthesis and biological evaluation of potential threonine synthase inhibitors: Rhizocticin A and Plumbemycin A.
Gahungu, Mathias; Arguelles-Arias, Anthony; Fickers, Patrick et al

in Bioorganic & Medicinal Chemistry (2013), 21(17), 4958-67

Rhizocticins and Plumbemycins are natural phosphonate antibiotics produced by the bacterial strains Bacillus subtilis ATCC 6633 and Streptomyces plumbeus, respectively. Up to now, these potential ... [more ▼]

Rhizocticins and Plumbemycins are natural phosphonate antibiotics produced by the bacterial strains Bacillus subtilis ATCC 6633 and Streptomyces plumbeus, respectively. Up to now, these potential threonine synthase inhibitors have only been synthesized under enzymatic catalysis. Here we report the chemical stereoselective synthesis of the non-proteinogenic (S,Z)-2-amino-5-phosphonopent-3-enoic acid [(S,Z)-APPA] and its use for the synthesis of Rhizocticin A and Plumbemycin A. In this work, (S,Z)-APPA was synthesized via the Still-Gennari olefination starting from Garner's aldehyde. The Michaelis-Arbuzov reaction was used to form the phosphorus-carbon bond. Oligopeptides were prepared using liquid phase peptide synthesis (LPPS) and were tested against selected bacteria and fungi. [less ▲]

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See detailLigand Binding Study of Human PEBP1/RKIP: Interaction with Nucleotides and Raf-1 Peptides Evidenced by NMR and Mass Spectrometry
Tavel, Laurette ULg; Jaquillard, Lucie; Karsisiotis, Andreas et al

in PLoS ONE (2012), 7(4): e36187

Background Human Phosphatidylethanolamine binding protein 1 (hPEBP1) also known as Raf kinase inhibitory protein (RKIP), affects various cellular processes, and is implicated in metastasis formation and ... [more ▼]

Background Human Phosphatidylethanolamine binding protein 1 (hPEBP1) also known as Raf kinase inhibitory protein (RKIP), affects various cellular processes, and is implicated in metastasis formation and Alzheimer's disease. Human PEBP1 has also been shown to inhibit the Raf/MEK/ERK pathway. Numerous reports concern various mammalian PEBP1 binding ligands. However, since PEBP1 proteins from many different species were investigated, drawing general conclusions regarding human PEBP1 binding properties is rather difficult. Moreover, the binding site of Raf-1 on hPEBP1 is still unknown. Methods/Findings In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands: GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants KD for different ligands. Native mass spectrometry was used as an alternative method for measuring KD values. Conclusions/Significance Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20°C to pH 7.5/NaCl 100 mM/30°C, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1. [less ▲]

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See detailAn alternative flexible conformation of the E. coli HUbeta(2) protein: structural, dynamics, and functional aspects.
Garnier, N.; Loth, K.; Coste, F. et al

in European Biophysics Journal [=EBJ] (2011), 40(2), 117-129

The histone-like HU protein is the major nucleoid-associated protein involved in the dynamics and structure of the bacterial chromosome. Under physiological conditions, the three possible dimeric forms of ... [more ▼]

The histone-like HU protein is the major nucleoid-associated protein involved in the dynamics and structure of the bacterial chromosome. Under physiological conditions, the three possible dimeric forms of the E. coli HU protein (EcHUalpha(2), EcHUbeta(2), and EcHUalphabeta) are in thermal equilibrium between two dimeric conformations (N(2) <--> I(2)) varying in their secondary structure content. High-temperature molecular dynamics simulations combined with NMR experiments provide information about structural and dynamics features at the atomic level for the N(2) to I(2) thermal transition of the EcHUbeta(2) homodimer. On the basis of these data, a realistic 3D model is proposed for the major I(2) conformation of EcHUbeta(2). This model is in agreement with previous experimental data. [less ▲]

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See detail1H, 13C and 15N backbone resonance assignments for the BS3 class A beta-lactamase from Bacillus licheniformis.
Vandenameele, Julie ULg; Matagne, André ULg; Damblon, Christian ULg

in Biomolecular NMR Assignments (2010), 4(2), 195-7

Class A beta-lactamases (260-280 amino acids; M ( r ) ~ 29,000) are among the largest proteins studied in term of their folding properties. They are composed of two structural domains: an all-alpha domain ... [more ▼]

Class A beta-lactamases (260-280 amino acids; M ( r ) ~ 29,000) are among the largest proteins studied in term of their folding properties. They are composed of two structural domains: an all-alpha domain formed by five to eight helices and an alpha/beta domain consisting of a five-stranded antiparallel beta-sheet covered by three to four alpha-helices. The alpha domain (~150 residues) is made up of the central part of the polypeptide chain whereas the alpha/beta domain (111-135 residues) is constituted by the N- and C-termini of the protein. Our goal is to determine in which order the different secondary structure elements are formed during the folding of BS3. With this aim, we will use pulse-labelling hydrogen/deuterium exchange experiments, in combination with 2D-NMR measurements, to monitor the time-course of formation and stabilization of secondary structure elements. Here we report the backbone resonance assignments as the requirement for further hydrogen/deuterium exchange studies. [less ▲]

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See detailNMR structure of a phosphatidylethanolamine binding protein from Drosophila
Rautureau, Gilles J P; Vovelle, Francoise; Schoentgen, Francoise et al

in Proteins (2010), 78(6), 1606-1610

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See detailHigh-level biosynthesis of the anteiso-C(17) isoform of the antibiotic mycosubtilin in Bacillus subtilis and characterization of its candidacidal activity.
Fickers, Patrick ULg; Guez, Jean-Sebastien; Damblon, Christian ULg et al

in Applied and Environmental Microbiology (2009), 75(13), 4636-40

High-level production (880 mg liter(-1)) and isolation of the anteiso-C(17) isoform of the lipopeptide mycosubtilin produced by a genetically engineered Bacillus subtilis strain are reported. Antifungal ... [more ▼]

High-level production (880 mg liter(-1)) and isolation of the anteiso-C(17) isoform of the lipopeptide mycosubtilin produced by a genetically engineered Bacillus subtilis strain are reported. Antifungal activity of this isoform, as determined via culture and fluorometric and cell leakage assays, suggests its potential therapeutic use as an antifungal agent, in particular against Candida spp. [less ▲]

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See detailPositively Cooperative Binding of Zinc Ions to Bacillus cereus 569/H/9 beta-Lactamase II Suggests that the Binuclear Enzyme Is the Only Relevant Form for Catalysis
Jacquin, Olivier ULg; Balbeur, Dorothée ULg; Damblon, Christian ULg et al

in Journal of Molecular Biology (2009), 392(5), 1278-1291

Metallo-beta-lactamases catalyze the hydrolysis of most beta-lactam antibiotics and hence represent a major clinical concern. While enzymes belonging to subclass B1 have been shown to display maximum ... [more ▼]

Metallo-beta-lactamases catalyze the hydrolysis of most beta-lactam antibiotics and hence represent a major clinical concern. While enzymes belonging to subclass B1 have been shown to display maximum activity as dizinc species, the actual metal-to-protein stoichiometry and the affinity for zinc are not clear. We have further investigated the process of metal binding to the beta-lactamase H from Bacillus cereus 569/H/9 (known as BcII). Zinc binding was monitored using complementary biophysical techniques, including circular dichroism in the far-UV, enzymatic activity measurements, competition with a chromophoric chelator, mass spectrometry, and nuclear magnetic resonance. Most noticeably, mass spectrometry and nuclear magnetic resonance experiments, together with catalytic activity measurements, demonstrate that two zinc ions bind cooperatively to the enzyme active site (with K-1/K-2 >= 5) and, hence, that catalysis is associated with the dizinc enzyme species only. Furthermore, competitive experiments with the chromophoric chelator Mag-Fura-2 indicates K-2 < 80 nM. This contrasts with cadmium binding, which is clearly a noncooperative process with the mono form being the only species significantly populated in the presence of 1 molar equivalent of Cd(II). Interestingly, optical measurements reveal that although the apo and dizinc species exhibit undistinguishable tertiary structural organizations, the metal-depleted enzyme shows a significant decrease in its alpha-helical content, presumably associated with enhanced flexibility. (C) 2009 Elsevier Ltd. All rights reserved. [less ▲]

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See detailPutative DNA G-quadruplex formation within the promoters of Plasmodium falciparum var genes
Smargiasso, Nicolas ULg; Gabelica, Valérie ULg; Damblon, Christian ULg et al

in BMC Genomics (2009), 10

Background. Guanine-rich nucleic acid sequences are capable of folding into an intramolecular four-stranded structure called a G-quadruplex. When found in gene promoter regions, G-quadruplexes can ... [more ▼]

Background. Guanine-rich nucleic acid sequences are capable of folding into an intramolecular four-stranded structure called a G-quadruplex. When found in gene promoter regions, G-quadruplexes can downregulate gene expression, possibly by blocking the transcriptional machinery. Here we have used a genome-wide bioinformatic approach to identify Putative G-Quadruplex Sequences (PQS) in the Plasmodium falciparum genome, along with biophysical techniques to examine the physiological stability of P. falciparum PQS in vitro. Results. We identified 63 PQS in the non-telomeric regions of the P. falciparum clone 3D7. Interestingly, 16 of these PQS occurred in the upstream region of a subset of the P. falciparum var genes (group B var genes). The var gene family encodes PfEMP1, the parasite’s major variant antigen and adhesin expressed at the surface of infected erythrocytes, that plays a key role in malaria pathogenesis and immune evasion. The ability of the PQS found in the upstream regions of group B var genes (UpsB-Q) to form stable Gquadruplex structures in vitro was confirmed using 1H NMR, circular dichroism, UV spectroscopy, and thermal denaturation experiments. Moreover, the synthetic compound BOQ1 that shows a higher affinity for DNA forming quadruplex rather than duplex structures was found to bind with high affinity to the UpsB-Q. Conclusions. This is the first demonstration of non-telomeric PQS in the genome of P. falciparum that form stable G-quadruplexes under physiological conditions in vitro. These results allow the generation of a novel hypothesis that the G-quadruplex sequences in the upstream regions of var genes have the potential to play a role in the transcriptional control of this major virulence-associated multi-gene family. [less ▲]

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See detailE. coli HU dimers dynamics studied by NMR
Augustyniak, R.; Damblon, Christian ULg; Castaing, B.

Poster (2008)

HU is a 2x90 residues, dimeric bacterial histone-like protein involved in chromosome compaction and other DNA-related processes. E. coli HU proteins are composed of three forms: two homodimers α2 and β2 ... [more ▼]

HU is a 2x90 residues, dimeric bacterial histone-like protein involved in chromosome compaction and other DNA-related processes. E. coli HU proteins are composed of three forms: two homodimers α2 and β2 (α and β sharing 70% of sequence identity) and one heterodimer (αβ). Dimeric forms relative abundance varies during cell growth and in response to environmental changes, suggesting that each dimer plays different physiological roles. Unlike other HU proteins which melt through a single step (N2<=>2D), E. coli dimers melt according to a two-step mechanism (N2<=>I2<=>2D). The native dimer, N2, melts partially into a dimeric intermediate, I2, which in turn yields the unfolded monomers, D. Circular Dichroism studies indicate that the intermediate, I2, corresponds to an HU dimer having partly lost its a-helices. Here we compared dynamic properties and structural features of E. Coli HU dimers at different temperatures in order to determine the secondary strutural elements still present in the I2 intermediate. Isotopically labelled proteins (15N, 13C) have been used to perform backbone assignment for all dimeric forms using sequential triple-resonance experiments. 15N-HSQC at different temperatures (288-323K) were recorded .Intensity variations and chemical shift perturbations allow the identification of the secondary element lost in the intermediate I2 confirming the high dynamics of arms responsible for protein-DNA interactions and the thermal stability of HTH motif in all dimeric forms. [less ▲]

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See detailStructural basis for the broad-spectrum inhibition of metallo-beta-lactamases by thiols
Lienard, Benoit M R; Garau, Gianpiero; Horsfall, Louise et al

in Organic & Biomolecular Chemistry (2008), 6(13), 2282-2294

The development of broad-spectrum metallo-beta-lactamase (MBL) inhibitors is challenging due to structural diversity and differences in metal utilisation by these enzymes. Analysis of structural data ... [more ▼]

The development of broad-spectrum metallo-beta-lactamase (MBL) inhibitors is challenging due to structural diversity and differences in metal utilisation by these enzymes. Analysis of structural data, followed by non-denturing mass spectrometric analyses, identified thiols proposed to inhibit representative MBLs from all three sub-classes: B1, B2 and B3. Solution analyses led to the identification of broad spectrum inhibitors, including potent inhibitors of the CphA MBL (Aeromonas hydrophila). Structural studies revealed that, as observed for other B1 and B3 MBLs, inhibition of the L1 MBL thiols involves metal chelation. Evidence is reported that this is not the case for inhibition of the CphA enzyme by some thiols; the crystal structure of the CphA-Zn-inhibitor complex reveals a binding mode in which the thiol does not interact with the zinc. The structural data enabled the design and the production of further more potent inhibitors. Overall the results suggest that the development of reasonably broad-spectrum MBL inhibitors should be possible. [less ▲]

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See detailA minimalistic approach to identify substrate binding features in B1 Metallo-beta-lactamases
Poeylaut-Palena, Andres A; Tomatis, Pablo E; Karsisiotis, Andreas I et al

in Bioorganic & Medicinal Chemistry Letters (2007), 17(18), 5171-5174

The 2-oxoazetidinylacetate sodium salt was synthesized as a model of a minimal P-lactam drug. This compound and the monobactam aztreonam were assayed as substrates of the Metallo-p-lactamase Bell. None of ... [more ▼]

The 2-oxoazetidinylacetate sodium salt was synthesized as a model of a minimal P-lactam drug. This compound and the monobactam aztreonam were assayed as substrates of the Metallo-p-lactamase Bell. None of them was hydrolyzed by the enzyme. While the azetidinone was not able to bind Bell, aztreonam was shown to bind in a nonproductive mode. These results provide an explanation for the unability of Metallo-beta-lactamases to inactive monobactams and give some clues for inhibitor design. (c) 2007 Elsevier Ltd. All rights reserved. [less ▲]

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See detailThe activity of the dinuclear cobalt-beta-lactamase from Bacillus cereus in catalysing the hydrolysis of beta-lactams
Badarau, Adriana; Damblon, Christian ULg; Page, Michael I

in Biochemical Journal (2007), 401(Part 1), 197-203

Metallo-beta-lactamases are native zinc enzymes that catalyse the hydrolysis of beta-lactam antibiotics, but are also able to function with cobalt(II) and require one or two nnetal-ions for catalytic ... [more ▼]

Metallo-beta-lactamases are native zinc enzymes that catalyse the hydrolysis of beta-lactam antibiotics, but are also able to function with cobalt(II) and require one or two nnetal-ions for catalytic activity. The hydrolysis of cefoxitin, cephaloridine and benzylpenicillin catalysed by CoBcII (cobalt-substituted beta-lactamase from Bacillus cereus) has been studied at different pHs and metal-ion concentrations. An enzyme group of pK(a) 6.52 +/- 0.1 is found to be required in its deprotionated form for metal-ion binding and catalysis. The species that results from the loss of one cobalt ion from the enzyme has no significant catalytic activity and is thought to be the mononuclear CoBcII. It appears that dinuclear CoBcII is the active form of the enzyme necessary for turnover, while the mononuclear CoBcII is only involved in substrate binding. The cobalt-substituted enzyme is a more efficient catalyst than the native enzyme for the hydrolysis of some beta-lactam antibiotics suggesting that the role of the metal-ion is predominantly to provide the nucleophilic hydroxide, rather than to act as a Lewis acid to polarize the carbonyl group and stabilize the oxyanion tetrahedral intermediate. [less ▲]

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See detailInhibitors of metallo-beta-lactamase generated from beta-lactam antibiotics.
Badarau, Adriana; Llinas, Antonio; Laws, Andrew P et al

in Biochemistry (2005), 44(24), 8578-89

The resistance of bacteria to the normally lethal action of beta-lactam antibiotics is largely due to the production of beta-lactamases that catalyze the hydrolysis of the beta-lactam. One class of these ... [more ▼]

The resistance of bacteria to the normally lethal action of beta-lactam antibiotics is largely due to the production of beta-lactamases that catalyze the hydrolysis of the beta-lactam. One class of these enzymes is a zinc-dependent metallo-beta-lactamase for which there are no clinically available inhibitors. The hydrolysis of cephalosporin beta-lactam antibiotics generates dihydrothiazines which subsequently undergo isomerization at C6 by C-S bond cleavage and through the intermediacy of a thiol. These thiols can be trapped by the beta-lactamase from Bacillus cereus, causing inhibition of the enzyme. The rate of production of the thiol corresponds to the rate of inhibition, and the inhibition constants are in the micromolar range but vary with the nature of the cephalosporin derivative. NMR studies have identified the structure of the thiols causing inhibition and also show that the thiol binds to the zinc ion, which in turn perturbs the metal-bound histidines. Inhibition is slowly removed as the thiol becomes oxidized or undergoes further degradation. The thiol intermediate generated from cephalothin is a slow binding inhibitor. There is no observed inhibition from the analogous degradation products from penicillins. [less ▲]

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See detailSynthesis of a simplified bryostatin C-ring analogue that binds to the CRD2 of human PKC-alpha and construction of a novel BC-analogue by an unusual Julia olefination process
Hale, K. J.; Frigerio, M.; Manaviazar, S. et al

in Organic Letters (2003), 5(4), 499-502

[GRAPHICS] The synthesis of two truncated bryostatin analogues 2 and 3 is described. High-field NMR measurements on the C-ring analogue 3 in (CH3CN)-H-2 containing 25% (H2O)-H-2 have shown that it binds ... [more ▼]

[GRAPHICS] The synthesis of two truncated bryostatin analogues 2 and 3 is described. High-field NMR measurements on the C-ring analogue 3 in (CH3CN)-H-2 containing 25% (H2O)-H-2 have shown that it binds to the CRD2 of human PKC-alpha at virtually the same position as phorbol-13-acetate (PA) and bryostatin 1 (1). NMR titration studies have also revealed that 3 binds to the CRD2 with a potency similar in magnitude to PA but much less potently than 1. [less ▲]

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See detailThe inhibitor thiomandelic acid binds to both metal ions in metallo-beta-lactamase and induces positive cooperativity in metal binding.
Damblon, Christian ULg; Jensen, Mikael; Ababou, Abdessamad et al

in Journal of Biological Chemistry (2003), 278(31), 29240-51

Thiomandelic acid is a simple, broad spectrum, and reasonably potent inhibitor of metallo-beta-lactamases, enzymes that mediate resistance to beta-lactam antibiotics. We report studies by NMR and ... [more ▼]

Thiomandelic acid is a simple, broad spectrum, and reasonably potent inhibitor of metallo-beta-lactamases, enzymes that mediate resistance to beta-lactam antibiotics. We report studies by NMR and perturbed angular correlation (PAC) spectroscopy of the mode of binding of the R and S enantiomers of thiomandelic acid, focusing on their interaction with the two metal ions in cadmium-substituted Bacillus cereus metallo-beta-lactamase. The 113Cd resonances are specifically assigned to the metals in the two individual sites on the protein by using 113Cd-edited 1H NMR spectra. Each enantiomer of thiomandelate produces large downfield shifts of both 113Cd resonances and changes in the PAC spectra, which indicate that they bind such that the thiol of the inhibitor bridges between the two metals. For R-thiomandelate, this is unambiguously confirmed by the observation of scalar coupling between Halpha of the inhibitor and both cadmium ions. The NMR and PAC spectra reveal that the two chiral forms of the inhibitor differ in the details of their coordination geometry. The complex with R-thiomandelate, but not that with the S-enantiomer, shows evidence in the PAC spectra of a dynamic process in the nanosecond time regime, the possible nature of which is discussed. The thiomandelate complex of the mononuclear enzyme can be detected only at low metal to enzyme stoichiometry; the relative populations of mononuclear and binuclear enzyme as a function of cadmium concentration provide clear evidence for positive cooperativity in metal ion binding in the presence of the inhibitor, in contrast to the negative cooperativity observed in the free enzyme. [less ▲]

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See detailDynamics of mononuclear cadmium beta-lactamase revealed by the combination of NMR and PAC spectroscopy.
Hemmingsen, L.; Damblon, Christian ULg; Antony, J. et al

in Journal of the American Chemical Society (2001), 123(42), 10329-35

The two metal sites in cadmium substituted beta-lactamase from Bacillus cereus 569/H/9 have been studied by NMR spectroscopy ((1)H, (15)N, and (113)Cd) and PAC spectroscopy ((111m)Cd). Distinct NMR ... [more ▼]

The two metal sites in cadmium substituted beta-lactamase from Bacillus cereus 569/H/9 have been studied by NMR spectroscopy ((1)H, (15)N, and (113)Cd) and PAC spectroscopy ((111m)Cd). Distinct NMR signals from the backbone amides are identified for the apoenzyme and the mononuclear and binuclear cadmium enzymes. For the binuclear cadmium enzyme, two (113)Cd NMR signals (142 and 262 ppm) and two (111m)Cd PAC nuclear quadrupole interactions are observed. Two nuclear quadrupole interactions are also observed, with approximately equal occupancy, in the PAC spectra at cadmium/enzyme ratios < 1; these are different from those derived for the binuclear cadmium enzyme, demonstrating interaction between the two metal ion binding sites. In contrast to the observation from PAC spectroscopy, only one (113)Cd NMR signal (176 ppm) is observed at cadmium/enzyme ratios < 1. The titration of the metal site imidazole (N)H proton signals as a function of cadmium ion-to-enzyme ratio shows that signals characteristic for the binuclear cadmium enzyme appear when the cadmium ion-to-enzyme ratio is between 1 and 2, whereas no signals are observed at stoichiometries less than 1. The simplest explanation consistent with all data is that, at cadmium/enzyme ratios < 1, the single Cd(II) is undergoing exchange between the two metal sites on the enzyme. This exchange must be fast on the (113)Cd NMR time scale and slow on the (111m)Cd PAC time scale and must thus occur in a time regime between 0.1 and 10 micros. [less ▲]

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