References of "Damblon, Christian"
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See detailThiomandelic acid, a broad spectrum inhibitor of zinc beta-lactamases: kinetic and spectroscopic studies.
Mollard, C.; Moali, C.; Papamicael, C. et al

in Journal of Biological Chemistry (2001), 276(48), 45015-23

Resistance to beta-lactam antibiotics mediated by metallo-beta-lactamases is an increasingly worrying clinical problem. Candidate inhibitors include mercaptocarboxylic acids, and we report studies of a ... [more ▼]

Resistance to beta-lactam antibiotics mediated by metallo-beta-lactamases is an increasingly worrying clinical problem. Candidate inhibitors include mercaptocarboxylic acids, and we report studies of a simple such compound, thiomandelic acid. A series of 35 analogues were synthesized and examined as metallo-beta-lactamase inhibitors. The K(i) values (Bacillus cereus enzyme) are 0.09 microm for R-thiomandelic acid and 1.28 microm for the S-isomer. Structure-activity relationships show that the thiol is essential for activity and the carboxylate increases potency; the affinity is greatest when these groups are close together. Thioesters of thiomandelic acid are substrates for the enzyme, liberating thiomandelic acid, suggesting a starting point for the design of "pro-drugs." Importantly, thiomandelic acid is a broad spectrum inhibitor of metallo-beta-lactamases, with a submicromolar K(i) value for all nine enzymes tested, except the Aeromonas hydrophila enzyme; such a wide spectrum of activity is unprecedented. The binding of thiomandelic acid to the B. cereus enzyme was studied by NMR; the results are consistent with the idea that the inhibitor thiol binds to both zinc ions, while its carboxylate binds to Arg(91). Amide chemical shift perturbations for residues 30-40 (the beta(3)-beta(4) loop) suggest that this small inhibitor induces a movement of this loop of the kind seen for other larger inhibitors. [less ▲]

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See detailDNA-binding mechanism of the Escherichia coli Ada O(6)-alkylguanine-DNA alkyltransferase.
Verdemato, P. E.; Brannigan, J. A.; Damblon, Christian ULg et al

in Nucleic Acids Research (2000), 28(19), 3710-8

The C-terminal domain of the Escherichia coli Ada protein (Ada-C) aids in the maintenance of genomic integrity by efficiently repairing pre-mutagenic O:(6)-alkylguanine lesions in DNA. Structural and ... [more ▼]

The C-terminal domain of the Escherichia coli Ada protein (Ada-C) aids in the maintenance of genomic integrity by efficiently repairing pre-mutagenic O:(6)-alkylguanine lesions in DNA. Structural and thermodynamic studies were carried out to obtain a model of the DNA-binding process. Nuclear magnetic resonance (NMR) studies map the DNA-binding site to helix 5, and a loop region (residues 151-160) which form the recognition helix and the 'wing' of a helix-turn-wing motif, respectively. The NMR data also suggest the absence of a large conformational change in the protein upon binding to DNA. Hence, an O:(6)-methylguanine (O:(6)meG) lesion would be inaccessible to active site nucleophile Cys146 if the modified base remained stacked within the DNA duplex. The experimentally determined DNA-binding face of Ada-C was used in combination with homology modelling, based on the catabolite activator protein, and the accepted base-flipping mechanism, to construct a model of how Ada-C binds to DNA in a productive manner. To complement the structural studies, thermodynamic data were obtained which demonstrate that binding to unmethylated DNA was entropically driven, whilst the demethylation reaction provoked an exothermic heat change. Methylation of Cys146 leads to a loss of structural integrity of the DNA-binding subdomain. [less ▲]

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See detailH-1-N-15 HMQC for the identification of metal-bound histidines in Cd-113-substituted Bacillus cereus zinc beta-lactamase
Damblon, Christian ULg; Prosperi, Christelle ULg; Lian, L. Y. et al

in Journal of the American Chemical Society (1999), 121(49), 11575-11576

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See detailThe catalytic mechanism of beta-lactamases: NMR titration of an active-site lysine residue of the TEM-1 enzyme.
Damblon, Christian ULg; Raquet, X.; Lian, L. Y. et al

in Proceedings of the National Academy of Sciences of the United States of America (1996), 93(5), 1747-52

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 ... [more ▼]

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. [less ▲]

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See detailStreptomyces K15 active-site serine DD-transpeptidase: specificity profile for peptide, thiol ester and ester carbonyl donors and pathways of the transfer reactions.
Grandchamps, Jacqueline; Nguyen-Distèche, Martine ULg; Damblon, Christian ULg et al

in Biochemical Journal (1995), 307(Pt 2), 335-339

The Streptomyces K15 transferase is a penicillin-binding protein presumed to be involved in bacterial wall peptidoglycan crosslinking. It catalyses cleavage of the peptide, thiol ester or ester bond of ... [more ▼]

The Streptomyces K15 transferase is a penicillin-binding protein presumed to be involved in bacterial wall peptidoglycan crosslinking. It catalyses cleavage of the peptide, thiol ester or ester bond of carbonyl donors Z-R1-CONH-CHR2-COX-CHR3-COO- (where X is NH, S or O) and transfers the electrophilic group Z-R1-CONH-CHR2-CO to amino acceptors via an acyl-enzyme intermediate. Kinetic data suggest that the amino acceptor behaves as a simple alternative nucleophile at the level of the acyl-enzyme in the case of thiol ester and ester donors, and that it binds to the enzyme.carbonyl donor Michaelis complex and influences the rate of enzyme acylation by the carbonyl donor in the case of amide donors. Depending on the nature of the scissile bond, the enzyme has different requirements for substituents at positions R1, R2 and R3. [less ▲]

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See detailThiolester substrates of DD-peptidases and beta-lactamases
Damblon, Christian ULg; Ledent, P.; Zhao, G. H. et al

in Letters In Peptide Science (1995), 2(3-4), 212-216

With peptide substrates, the penicillin-sensitive DD-peptidases exhibit a strict specificity for D-Ala-D-Xaa C-termini. Only glycine is tolerated as the C-terminal residue, but with a significantly ... [more ▼]

With peptide substrates, the penicillin-sensitive DD-peptidases exhibit a strict specificity for D-Ala-D-Xaa C-termini. Only glycine is tolerated as the C-terminal residue, but with a significantly decreased activity. These enzymes also hydrolyse various ester and thiolester analogues of their natural substrates. Some of the thiolesters whose C-terminal leaving group exhibited an L stereochemistry were significantly hydrolysed by some of the studied enzymes, particularly by the Actinomadura R39 DD-peptidase. By contrast, the strict specificity for a D residue in the penultimate position was fully retained. The same esters and thiolesters also behaved as substrates for beta-lactamases. In this case, thiolesters exhibiting L stereochemistry in the C-terminal position could also be hydrolysed, mainly by the class C and class D enzymes. But, more surprisingly, the class C Enterobacter cloacae P99 beta-lactamase also hydrolysed thiolesters containing an L residue in the penultimate position, sometimes more efficiently than the D isomer. [less ▲]

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See detailBreakdown of the stereospecificity of DD-peptidases and beta-lactamases with thiolester substrates.
Damblon, Christian ULg; Zhao, G. H.; Jamin, M. et al

in Biochemical Journal (1995), 309 ( Pt 2)

With peptide analogues of their natural substrates (the glycopeptide units of nascent peptidoglycan), the DD-peptidases exhibit a strict preference for D-Ala-D-Xaa C-termini. Gly is tolerated as the C ... [more ▼]

With peptide analogues of their natural substrates (the glycopeptide units of nascent peptidoglycan), the DD-peptidases exhibit a strict preference for D-Ala-D-Xaa C-termini. Gly is tolerated as the C-terminal residue, but with a significantly decreased activity. These enzymes were also known to hydrolyse various ester and thiolester analogues of their natural substrates. Some thiolesters with a C-terminal leaving group that exhibited L stereochemistry were significantly hydrolysed by some of the enzymes, particularly the Actinomadura R39 DD-peptidase, but the strict specificity for a D residue in the penultimate position was fully retained. These esters and thiolesters also behave as substrates for beta-lactamases. In this case, thiolesters exhibiting L stereochemistry in the ultimate position could also be hydrolysed, mainly by the class-C and class-D enzymes. However, more surprisingly, the class-C Enterobacter cloacae P99 beta-lactamase also hydrolysed thiolesters containing an L residue in the penultimate position, sometimes with a higher efficiency than the D isomer. [less ▲]

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See detailSynthesis, purification and kinetic properties of fluorescein-labelled penicillins
Lakaye, Bernard ULg; Damblon, Christian ULg; Jamin, Marc et al

in Biochemical Journal (1994), 300

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See detailDirect n.m.r. evidence for substrate-induced conformational changes in a beta-lactamase.
Jamin, M.; Damblon, Christian ULg; Bauduin-Misselyn, A. M. et al

in Biochemical Journal (1994), 301 ( Pt 1)

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 ... [more ▼]

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. [less ▲]

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See detailThe Mechanism of Action of DD-Peptidases: The Role of Tyrosine-159 in the Streptomyces R61 DD-Peptidase
Wilkin, Jean-Marc; Jamin, Marc; Damblon, Christian ULg et al

in Biochemical Journal (1993), 291(Part 2), 537-544

Tyrosine-159 of the Streptomyces R61 penicillin-sensitive DD-peptidase was replaced by serine or phenylalanine. The second mutation yielded a very poorly active protein whose rate of penicillin binding ... [more ▼]

Tyrosine-159 of the Streptomyces R61 penicillin-sensitive DD-peptidase was replaced by serine or phenylalanine. The second mutation yielded a very poorly active protein whose rate of penicillin binding was also drastically decreased, except for the reactions with nitrocefin and methicillin. The consequences of the first mutation were more surprising, since a large proportion of the thiolesterase activity was retained, together with the penicillin-binding capacity. Conversely, the peptidase properties was severely affected. In both cases, a drastic decrease in the transferase activity was observed. The results are compared with those obtained by mutation of the corresponding residue in the class A beta-lactamase of Streptomyces albus G. [less ▲]

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See detailPENICILLIN-BINDING PROTEIN 2X OF STREPTOCOCCUS-PNEUMONIAE - ENZYMATIC-ACTIVITIES AND INTERACTIONS WITH BETA-LACTAMS
JAMIN, M.; Damblon, Christian ULg; MILLIER, S. et al

in Biochemical Journal (1993), 292(Part 3), 735-741

The high-molecular-mass penicillin-binding protein (PBP) 2x, one of the primary targets of beta-lactam antibiotics in Streptococcus pneumoniae, has been produced as a soluble form and purified in large ... [more ▼]

The high-molecular-mass penicillin-binding protein (PBP) 2x, one of the primary targets of beta-lactam antibiotics in Streptococcus pneumoniae, has been produced as a soluble form and purified in large amounts. It has been shown to catalyse hydrolysis and transfer reactions with different ester and thiolester substrates and its catalytic behaviour was often similar to that of the soluble DD-peptidase from Streptomyces R61. This provided an easy method to monitor the activity of the PBP. For the first time, a reliable kinetic study of the interaction between a lethal target and beta-lactam antibiotics has been performed. Characteristic kinetic parameters were obtained with different beta-lactam compounds. These results not only validated the mechanism established with non-essential extracellular enzymes, but will also constitute the basis for comparative studies of the low-affinity variants from penicillin-resistant strains. [less ▲]

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See detailMechanism of action of β-lactamases and DD-peptidases
Frère, Jean-Marie ULg; Joris, Bernard ULg; Jacob, Françoise et al

in Pandit, U.K.; Alderweireldt, F.C. (Eds.) Bioorganic Chemistry in Healthcare and Technology (1991)

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See detailTHE MUTATION LYS234HIS YIELDS A CLASS-A BETA-LACTAMASE WITH A NOVEL PH-DEPENDENCE
BRANNIGAN, J.; Matagne, André ULg; Jacob, Françoise et al

in Biochemical Journal (1991), 278(Part 3), 673-678

The lysine-234 residue is highly conserved in beta-lactamases and in nearly all active-site-serine penicillin-recognizing enzymes. Its replacement by a histidine residue in the Streptomyces albus G class ... [more ▼]

The lysine-234 residue is highly conserved in beta-lactamases and in nearly all active-site-serine penicillin-recognizing enzymes. Its replacement by a histidine residue in the Streptomyces albus G class A beta-lactamase yielded an enzyme the pH-dependence of which was characterized by the appearance of a novel pK, which could be attributed to the newly introduced residue. At low pH, the k(cat.) value for benzylpenicillin was as high as 50 % of that of the wild-type enzyme, demonstrating that an efficient active site was maintained. Both k(cat.) and k(cat.)/K(m) dramatically decreased above pH 6 but the decrease in k(cat.)/K(m) could not be attributed to larger K(m) values. Thus a positive charge on the side chain of residue 234 appears to be more essential for transition-state stabilization than for initial recognition of the substrate ground state. [less ▲]

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See detailACCUMULATION OF ACYL-ENZYME IN DD-PEPTIDASE-CATALYZED REACTIONS WITH ANALOGS OF PEPTIDE-SUBSTRATES
JAMIN, M.; Adam, Maggy; Damblon, Christian ULg et al

in Biochemical Journal (1991), 280(Part 2), 499-506

Thioester substrates can be used to study the hydrolysis and transfer reactions catalysed by beta-lactamases and DD-peptidases. With the latter enzymes, accumulation of the acyl-enzyme can be detected ... [more ▼]

Thioester substrates can be used to study the hydrolysis and transfer reactions catalysed by beta-lactamases and DD-peptidases. With the latter enzymes, accumulation of the acyl-enzyme can be detected directly. The efficiency of various amines as acceptor substrates was in excellent agreement with previous results obtained with peptide substrates of the DD-peptidases. The results indicated the presence of a specific binding site for the acceptor substrates, Although most of the results agreed well with a simple partition model, more elaborate hypotheses will be needed to account for all the data presented. [less ▲]

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See detailAcyltransferase activities of the high-molecular-mass essential penicillin-binding proteins
Adam, Maggy; Damblon, Christian ULg; Jamin, Marc et al

in Biochemical Journal (1991), 279(Part 2), 601-604

The high-molecular-mass penicillin-binding proteins (HMM-PBPs), present in the cytoplasmic membranes of all eubacteria, are involved in important physiological events such as cell elongation, septation or ... [more ▼]

The high-molecular-mass penicillin-binding proteins (HMM-PBPs), present in the cytoplasmic membranes of all eubacteria, are involved in important physiological events such as cell elongation, septation or shape determination. Up to now it has, however, been very difficult or impossible to study the catalytic properties of the HMM-PBPs in vitro. With simple substrates, we could demonstrate that several of these proteins could catalyse the hydrolysis of some thioesters or the transfer of their acyl moiety on the amino group of a suitable acceptor nucleophile. Many of the acyl-donor substrates were hippuric acid or benzoyl-D-alanine derivatives, and their spectroscopic properties enabled a direct monitoring of the enzymic reaction. In their presence, the binding of radioactive penicillin to the PBPs was also inhibited. [less ▲]

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See detailCHROMOGENIC DEPSIPEPTIDE SUBSTRATES FOR BETA-LACTAMASES AND PENICILLIN-SENSITIVE DD-PEPTIDASES
Adam, M.; Damblon, Christian ULg; PLAITIN, B. et al

in Biochemical Journal (1990), 270(2), 525-529

Various ester and thioester derivatives of hippuric acid have been prepared which were substrates of both beta-lactamases and DD-peptidases. The thioesters were more rapidly hydrolysed by nearly all the ... [more ▼]

Various ester and thioester derivatives of hippuric acid have been prepared which were substrates of both beta-lactamases and DD-peptidases. The thioesters were more rapidly hydrolysed by nearly all the enzymes. Surprisingly, the enzymes acted rather efficiently on substrates which did not contain any chiral centre. [less ▲]

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