References of "Ghuysen, Jean-Marie"
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See detailThe penicillin-binding proteins in Streptococcus faecalis ATCC 9790
Coyette, Jean; Ghuysen, Jean-Marie ULg; Fontana, Roberta

in European Journal of Biochemistry (1980), 110(2), 445-456

Streptococcus faecalis ATCC 9790 possesses seven membrane-bound penicillin-binding proteins. They have been characterized with respect to their apparent molecular weights, relative abundance, specificity ... [more ▼]

Streptococcus faecalis ATCC 9790 possesses seven membrane-bound penicillin-binding proteins. They have been characterized with respect to their apparent molecular weights, relative abundance, specificity profiles for 15 different beta-lactam antibiotics and stability under various conditions. In water and at 37 degrees C, all the native penicillin-binding proteins have half-lives longer than 20 h except protein 3b (half-life of about 600 min) and protein 4 (half-life of about 175 min). The short-lived 80 000-Mr protein 4 is spontaneously converted into a 73 000-Mr water-soluble, penicillin-binding protein 4. Similarly, the short-lived 82 000-Mr protein 3b seems to be the protein from which the 72 000-Mr water-soluble protein X spontaneously originates during incubation of the membranes. Release of both proteins 4 and X from the membrane is maximal under alkaline conditions; it is not inhibited by various protease inhibitors. After exposure to trypsin, the 43 000-Mr membrane-bound penicillin binding protein 6 (a DD-carboxypeptidase) gives to a 30 000-Mr water-soluble protein 6. Like the parent protein, protein 6 exhibits both DD-carboxypeptidase activity and penicillin-binding ability. With proteins 6 and 6, low dose levels of p-chloromercuribenzoate prevent both enzyme activity and combination with penicillin, thus strongly suggesting that a thiol group is involved in the enzyme active center. We have shown previously [Coyette et al. in Eur. J. Biochem. 88, 297--305 (1978) and 75, 231--239 (1977)] that the DD-carboxypeptidase protein 6 fragments the benzylpenicillin molecule with formation of phenylacetylglycine. Breakdown of the complex formed between [14C]benzylpenicillin and 14 000-Mr membrane-bound protein 1 is also 'enzyme-catalysed'. Most likely, however, the released product is penicilloate. With all the other penicillin-binding proteins whose molecular weights are intermediate between those of proteins 1 and 6, breakdown of the complexes formed with [14C]benzylpenicillin results from proteolysis and is not due to the release of the bound metabolite. None of the penicillin-binding proteins behaves, by itself, as a lethal target for beta-lactam antibiotic action on the living cells. [less ▲]

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See detailThe exocellular DD-carboxypeptidase of Streptomyces albus G: A metallo (Zn2+) enzyme
Dideberg, O.; Joris, Bernard ULg; Frère, Jean-Marie ULg et al

in FEBS Letters (1980), 117(1-2), 215-218

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See detailThe active centres in penicillin-sensitive enzymes
Ghuysen, Jean-Marie ULg; Frère, Jean-Marie ULg; Leyh-Bouille, Mélina et al

in Philosophical Transactions : Biological Sciences (1980), 289(1036), 285-301

The interaction between beta-lactam antibiotics and the penicillin-sensitive enzymes is a multiple-step process. Binding of the beta-lactam ring of the penam (or 3-cepham) nucleus occurs at binding site ... [more ▼]

The interaction between beta-lactam antibiotics and the penicillin-sensitive enzymes is a multiple-step process. Binding of the beta-lactam ring of the penam (or 3-cepham) nucleus occurs at binding site no. 1. Interaction between the N-14 substituent of the bound molecule and binding site no. 2 induces changes in binding site no. 1. In turn, the catalytic site thus created increases the chemical reactivity of the beta-lactam amide bond. As the beta-lactam ring opens and acylates an enzyme serine residue, the interaction between the thiazolidine (or dihydrothiazine) ring and binding site no. 3 stabilizes the acyl-enzyme complex. Enzyme regeneration slowly proceeds either by direct elimination of the penicilloyl moiety or via C-5-C-6 splitting of the bound metabolite. The fragment arising from thiazolidine yields free N-formyl-D-penicillamine while the enzyme-linked N-acylglycyl fragment is immediately attacked by an exogenous nucleophile correctly positioned on the acceptor site. Similarly, the enzyme action on L-X-D-Ala-D-Ala terminated peptides is mediated via a binding site no. 1 that combines with D-Ala-D-Ala, a binding site no. 2 that interacts with the side chain of the preceding L-residue, an inducible catalytic site and an acceptor site. Enzymes are known that form a transitory L-X-D-Ala-enzyme complex where the acyl group is ester-linked to the same serine residue as that involved in the formation of the penicilloyl-enzyme complex (Waxman et al., this symposium). Other enzymes, however, may function as catalyst templates. Depending on the enzymes, the independence of the beta-lactam and L-X-D-Ala-D-Ala active centres is more or less pronounced. [less ▲]

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See detailPenicillin-binding proteins in the membranes of Streptomyces sp
Dusart, Jean; Leyh-Bouille, Mélina; Nguyen-Distèche, Martine ULg et al

in Archives Internationales de Physiologie et de Biochimie (1980, February 01), 88(1), 27-29

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See detailAntibiotics and peptidoglycan metabolism
Ghuysen, Jean-Marie ULg

in Sammes, Peter G (Ed.) Topics in Antibiotic Chemistry. vol 5 (1980)

A review with 305 refs. is given on bacterial cell wall peptidoglycan metab. and on the mechanisms of action of antibiotics that inhibit cell wall formation. [on SciFinder(R)]

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See detailThe 4.5 Å resolution structure analysis of the exocellular DD-carboxypeptidase of Streptomyces albus G.
Dideberg, Otto; Charlier, Paulette ULg; Dupont, Léon et al

in FEBS Letters (1980), 117(1), 212-214

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See detailOn the Active Centers of Serine and Zn II DD-carboxyppetidases
Charlier, Paulette ULg; Coyette, Jacques ULg; Dideberg, Otto et al

in Gregory, G.I. (Ed.) Recent advances in the Chemistry of beta-lactam antibiotics (1980)

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See detailCrystallographic data for the DD-carboxypeptidase-endopeptidase of low penicillin sensitivity excreted by Streptomyces albus g
Dideberg, Otto; Frère, Jean-Marie ULg; Ghuysen, Jean-Marie ULg

in Journal of Molecular Biology (1979), 129(4), 677-679

The DD-carboxypeptidase-endopeptidase of low penicillin sensitivity that is excreted by Streptomyces albus G has been crystallized from a polyethylene glycol (Mr 6000 to 7500) solution at pH 8.0. X-ray ... [more ▼]

The DD-carboxypeptidase-endopeptidase of low penicillin sensitivity that is excreted by Streptomyces albus G has been crystallized from a polyethylene glycol (Mr 6000 to 7500) solution at pH 8.0. X-ray examination of the prismatic crystals shows that the space group is P21 with unit cell dimensions a = 51.1 Å, B = 49.7 Å, C = 38.7 Å, β = 100.6 ° and one molecule in the asymmetric unit. A crystal suspension made in 50 mM -Tris • HCl buffer (pH 8.0) supplemented with 5 mM-MgCl2 and 16% (w/v) polyethylene glycol exhibits enzyme activity on the substrate Ac2-L-Lys-D-Ala-D-Ala. [less ▲]

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See detailEffects of nucleophiles on the breakdown of the benzylpenicilloyl-enzyme complex EI formed between benzylpenicillin and the exocellular DD-carboxypeptidase--transpeptiase of Streptomyces strain R61
Marquet, Alberto; Frère, Jean-Marie ULg; Ghuysen, Jean-Marie ULg et al

in Biochemical Journal (1979), 177(3), 909-916

Serine is one of the enzyme residues with which benzylpenicillin collides as a result of its binding to the Streptomyces strain-R61 DD-carboxypeptidase-transpeptidase enzyme. Nucleophilic attack occurs on ... [more ▼]

Serine is one of the enzyme residues with which benzylpenicillin collides as a result of its binding to the Streptomyces strain-R61 DD-carboxypeptidase-transpeptidase enzyme. Nucleophilic attack occurs on C(7) of the bound antibiotic molecule with formation of a benzylpenicilloyl-serine ester linkage, i.e. formation of the benzylpenicilloyl-enzyme EI complex. To reject the bound penicilloyl moiety and consequently to recover its initial activities, the strain-R61 enzyme has developed two possible mechanisms. Pathway A is a direct attack of the serine ester linkage by an exogenous nucleophile, resulting in the transfer of the benzylpenicilloyl moiety to this nucleophile. In pathway B, the benzylpenicilloyl moiety is first fragmented by C(5)-C(6) cleavage and the enzyme-bound phenylacetylglycyl residue thus produced is in turn transferred to the nucleophile. Pathway B occurs with water, glycylglycine and other amino compounds. Both pathways A and B occur with glycerol, other ROH nucleophiles and neutral hydroxylamine. The nucleophilic attacks are enzyme-catalysed. [less ▲]

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See detailNMR evidence for the structure of the complex between penicillin and the DD-carboxypeptidase of streptomyces R61
Degelaen, J.; Feeney, J.; Roberts, G. C. K. et al

in FEBS Letters (1979), 98(1), 53-56

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See detailCrystallographic data for a penicillin receptor : exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61
Knox, James R.; DeLucia, Mary L.; Murthy, N. S. et al

in Journal of Molecular Biology (1979), 127(2), 217-218

A pencillin-sensitive enzyme, the exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61, has been crystallized from polyethylene glycol (Mr = 6000 to 7500) solution at pH 7•6. X-ray ... [more ▼]

A pencillin-sensitive enzyme, the exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61, has been crystallized from polyethylene glycol (Mr = 6000 to 7500) solution at pH 7•6. X-ray examination of the orthorhombic crystals shows the space group is P212121, with unit cell dimensions a = 51•1 Å, B = 67•4 Å, and C = 102•9 Å. With four molecules of molecular weight 38,000, the Å3/dalton ratio for the cell is 2•33. The crystals are stable to irradiation for 75 hours and are suitable for structure analysis to at least 2•4 Å resolution. The radius of gyration of the molecule in solution at pH 6.8 is 20.8 Å. [less ▲]

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See detailUse of model enzymes in the determination of the mode of action of penicillins and delta 3-cephalosporins
Ghuysen, Jean-Marie ULg; Frère, Jean-Marie ULg; Leyh-Bouille, Mélina et al

in Annual Review of Biochemistry (1979), 48

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See detailInteraction between penicillin and its enzyme target
Ghuysen, Jean-Marie ULg; Frère, Jean-Marie ULg; Leyh-Bouille, Mélina et al

in Microbial Drug Resistance : Mechanism, Epidemiology, & Disease (1979), 2

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See detailSolubilization and isolation of the membrane-bound DD-carboxypeptidase of Streptococcus faecalis ATCC9790. Properties of the purified enzyme
Coyette, Jacques; Ghuysen, Jean-Marie ULg; Fontana, Roberta

in European Journal of Biochemistry (1978), 88(1), 297-305

Streptococcus faecalis ATCC 9790 possesses six membrane-bound, penicillin-binding proteins. That numbered 6 (Mr 43000) is the most abundant one and is the DD-carboxypeptidase studied previously. The ... [more ▼]

Streptococcus faecalis ATCC 9790 possesses six membrane-bound, penicillin-binding proteins. That numbered 6 (Mr 43000) is the most abundant one and is the DD-carboxypeptidase studied previously. The enzyme has been solubilized and purified to the stage where one single protein band can be detected by gel electrophoresis. The purification procedure does not alter the properties that the enzyme exhibits when it is membrane-bound. The DD-carboxypeptidase itself may be a killing target for penicillin in S. faecalis. [less ▲]

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See detailStability of D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid in relation to its possible occurrence as a degradation product of penicillin by the exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61 and the membrane-bound dd-carboxypeptidase from Bacillus stearothermophilus
Adriaens, Paul; Meesschaert, Boudewijn; Frère, Jean-Marie ULg et al

in Journal of Biological Chemistry (1978), 253(10), 3660-3665

The stability of D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid has been studied under various conditions. In 10 mM cacodylate, pH 6.5, and at 55 degrees C, D-5,5-dimethyl-delta2-thiazoline-4 ... [more ▼]

The stability of D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid has been studied under various conditions. In 10 mM cacodylate, pH 6.5, and at 55 degrees C, D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid (at concentrations lower than 1 mM) is hydrolyzed into N-formyl-D-penicillamine with a half-life of 3 to 4 min. On this basis, it is very unlikely that D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid could be one of the end products resulting from the cleavage of benzylpenicillin by the DD-carboxypeptidase of Bacillus stearothermophilus (as reported by Hammarstrom and Strominger (1976) J. Biol. Chem. 251, 7947--7949). In 3 mM phosphate, pH 7.5, and at 37 degrees C, D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid (at concentrations lower than 1 mM) has a half-life of 45 min. On the basis of kinetic experiments carried out under these conditions with phenoxymethylpenicillin and the DD-carboxypeptidase-transpeptidase of Streptomyces R61, it is concluded that the primary product which arises from the thiazolidine moiety of the antibiotic molecule and gives rise to N-formyl-D-penicillamine, has a half-life of 10 min, a value which is not compatible with the hypothesis that D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid would be an intermediate involved in the fragmentation pathway. [less ▲]

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See detailInteraction between penicillin and the DD-carboxypeptidase of the unstable L-form of Proteus mirabilis strain 19
Schilf, Wolfgang; Frere, Philippe; Frère, Jean-Marie ULg et al

in European Journal of Biochemistry (1978), 85(2), 325-330

Binding of penicillin to the DD-carboxypeptidase of the unstable spheroplast L-form of Proteus mirabilis results in the rapid formation of a modified enzyme-inhibitor complex which in turn undergoes rapid ... [more ▼]

Binding of penicillin to the DD-carboxypeptidase of the unstable spheroplast L-form of Proteus mirabilis results in the rapid formation of a modified enzyme-inhibitor complex which in turn undergoes rapid decay into reactivated enzyme and an antibiotically inactive penicillin degradation product. Major antibiotic metabolites recovered from such interactions were benzylpenicilloic acid and phenoxymethylpenicilloic acid from benzylpenicillin and phenoxymethylpenicillin, respectively, suggesting a second enzymic function of the DD-carboxypeptidase as a penicillinase of low efficiency. Statistical analyses made with the help of a linear regression program show that the enzyme interacts with the substrate UDP-N-acetylmuramoyl-L-alanyl-D-gamma-glutamyl-(L)-meso-2,6-diaminopimelyl -(L)-D-alanyl-D-alanine and either benzympenicillin or carbenicillin in a non-competitive manner. [less ▲]

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See detailLarge Scale Preparation of Purified Exocellular DD-carboxypeptidase-Transpeptidase of Streptomyces Strain R61
Fossati, P.; Saint-Ghislain, M.; Sicard, P. J. et al

in Biotechnology and Bioengineering (1978), 20

The exocellular DD-carboxypeptidase–transpeptidase that Streptomyces R61 excretes during growth has been produced in large fermentation units of 15 m3 total capacity. The yield from 15,000 liter culture ... [more ▼]

The exocellular DD-carboxypeptidase–transpeptidase that Streptomyces R61 excretes during growth has been produced in large fermentation units of 15 m3 total capacity. The yield from 15,000 liter culture filtrate was 1.080 g purified enzyme (92% purity) with a total recovery of 29% and at least a 2000-fold increased specific activity. [less ▲]

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See detailThe exocellular DD-carboxypeptidase-endopeptidase from Streptomyces albus Gl. Purification and chemical properties
Duez, Colette ULg; Frère, Jean-Marie ULg; Geurts, Francine et al

in Biochemical Journal (1978), 175(3), 793-800

The exocellular DD-carboxypeptidase-endopeptidase of Streptomyces albus G was purified to protein homogeneity and compared with the exocellular DD-carboxypeptidases-transpeptidases of Streptomyces R61 and ... [more ▼]

The exocellular DD-carboxypeptidase-endopeptidase of Streptomyces albus G was purified to protein homogeneity and compared with the exocellular DD-carboxypeptidases-transpeptidases of Streptomyces R61 and Actinomadura R39. The S. albus G enzyme, as it is isolated, occurs in two forms. Enzyme I (30% of the total amount) and enzyme II (70% of the total amount) are identical in all respects, except that, by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, enzyme I has an apparent mol. wt. (9000) that is half of that found by molecular-sieve filtration under non-denaturing conditions. Irrespective of the technique used, enzyme II has an apparent mol. wt. of about 18500. [less ▲]

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See detailThe peptidoglycan crosslinking enzyme system in Streptomyces strains R61, K15 and rimosus : Kinetic Coefficients Involved in the Interactions of the Membrane-Bound Transpeptidase with Peptide Substrates and β-Lactam Antibiotics
Dusart, Jean; Leyh-Bouille, Mélina; Ghuysen, Jean-Marie ULg

in European Journal of Biochemistry (1977), 81(1), 33-44

The transpeptidation reaction performed by the membranes of Streptomyces strain R61 fits the general rate equation for an enzyme-catalysed bimolecular reaction. The same membranes (E) interact with beta ... [more ▼]

The transpeptidation reaction performed by the membranes of Streptomyces strain R61 fits the general rate equation for an enzyme-catalysed bimolecular reaction. The same membranes (E) interact with beta-lactams (I) to form inactive penicillin-enzyme-membrane complexes (EI) of rather high stability, which subsequently break down (E + I leads to EI leads to E + degradation products). The enzyme is regenerated and the antibiotic is released in the form of an inactive metabolite. With benzylpenicillin, the degradation product is benzylpenicilloic acid. The reaction is heat-labile. The first step of the reaction (E + I leads to EI) is characterized by a second-order rate constant (kformation in M-1 s-1) and the second step (EI leads to E + degradation products) by a first-order rate constant (kbreakdown in s-1). The effects in vitro of various beta-lactams on the membrane-bound transpeptidase, as expressed by the relevant kformation and kbreakdown values, parallel the effects in vivo of the same antibiotics as expressed by their ability to prevent the germination and growth of conidiospores. The kinetic parameters of the transpeptidase that was solubilized with N-cetyl-N,N,N-trimethylammonium bromide with respect to its interaction with both peptide substrates and beta-lactam antibiotics are quantitatively different from those of the membrane-bound enzyme. Moreover, the solubilized enzyme fragments benzylpenicillin with formation of phenylacetylglycine, a reaction which is similar to that catalysed by the exocellular R61 enzyme. The membranes of Streptomyces strains rimosus and K15 possess an active 'classic' penicillinase. They were not studied but the kinetic coefficients of the corresponding solubilized transpeptidases were determined and compared with those of the solubilized enzyme from strain R61. [less ▲]

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