References of "Ghuysen, Jean-Marie"
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See detailThe catalytic activity and penicillin sensitivity in the liquid and frozen states of membrane-bound and detergent-solubilised transpeptidase of Streptomyces R61
Dusart, Jean; Marquet, Alberto; Ghuysen, Jean-Marie ULg et al

in European Journal of Biochemistry (1975), 56(1), 57-65

The Km, app. values of the membrane-bound transpeptidase of Streptomyces R61 for the donor Ac2-L-Lys-D-Ala-D-Ala and the acceptor Gly-Gly are not affected by temperature variations when the reaction ... [more ▼]

The Km, app. values of the membrane-bound transpeptidase of Streptomyces R61 for the donor Ac2-L-Lys-D-Ala-D-Ala and the acceptor Gly-Gly are not affected by temperature variations when the reaction mixtures are incubated in liquid suspensions. At -5 degrees C, the incubation can be carried out either in the liquid or in the frozen state. The enzyme is active in the latter state. In the frozen state, the Km, app. value for the acceptor remains unchanged but there is a 3-fold increase in the maximum velocity, a 10-fold decrease of the Km, app. value for the donor and a 10-fold increase of the benzylpenicillin concentration required to inhibit the enzyme activity by 50% (ID50 value). Temperatures of -35 degrees C or below are required to completely inhibit the membrane-bound enzyme in the frozen state. Cetyltrimethylammonium bromide extracts the transpeptidase both from the isolated membranes and, with a much higher yield, from the intact mycelium. The extracted enzyme is not active in the frozen state, requires detergent for activity, has decreased Km, app. values for both donor and acceptor, exhibits the same sensitivity to benzylpenicillin and cephalosporin C as the membrane-bound transpeptidase (in liquid suspensions) and, like this latter enzyme, has no DD-carboxypeptidase activity. The detergent-extracted transpeptidase penetrates gels of Sephadex-100 and is not sedimented at 200 000 X g. [less ▲]

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See detailA donor-acceptor substrate of the exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61
Zeiger, Allen R; Frère, Jean-Marie ULg; Ghuysen, Jean-Marie ULg et al

in FEBS Letters (1975), 52(2), 221-225

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See detailLa paroi bactérienne
Ghuysen, Jean-Marie ULg

in Gasser, François (Ed.) Bactéries, bactériophages : cours de l'Institut Pasteur (1975)

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See detailParoi bactérienne, membrane cytoplasmique et pénicilline
Ghuysen, Jean-Marie ULg

in Microbia (1975), A(2),

Bacterial transpeptidases specifically recognize peptide donor, peptide acceptor and β-lactam antibiotics. The identification of the chemical modifications undergone by the antibiotic during its ... [more ▼]

Bacterial transpeptidases specifically recognize peptide donor, peptide acceptor and β-lactam antibiotics. The identification of the chemical modifications undergone by the antibiotic during its interaction with the enzyme and the characterization of the enzyme centers involved in hydrolysis, transpeptidation and breakdown of the antibiotic molecule are the next problems which must be solved in order to fully understand how penicillin kills bacteria. [less ▲]

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See detailBeta-lactamases (Actinomycetes species)
Johnson, Kenneth; Duez, Colette ULg; Frère, Jean-Marie ULg et al

in Methods in Enzymology (1975), XLIII

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See detailInteraction between beta-lactam antibiotics and exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R61
Frère, Jean-Marie ULg; Leyh-Bouille, Mélina; Ghuysen, Jean-Marie ULg et al

in European Journal of Biochemistry (1974), 50(1), 203-214

On the basis of steady-state kinetics, inhibition of the exocellular dd-carboxypeptidase-trans-peptidase of Streptomyces R61 by β-lactam antibiotics was competitive with regard to the donor substrate ... [more ▼]

On the basis of steady-state kinetics, inhibition of the exocellular dd-carboxypeptidase-trans-peptidase of Streptomyces R61 by β-lactam antibiotics was competitive with regard to the donor substrate. However, the complexes formed between the Streptomyces R61 enzyme and various β-lactam antibiotics were relatively stable, exhibiting half-lives of 40 to 80 min at 37°C and neutral pH. During breakdown of the complexes the protein underwent reactivation, whereas the released antibiotic molecule was chemically altered. With [14C]benzylpenicillin, the released compound was neither benzylpenicillin nor benzylpenicilloic acid. The properties of the Streptomyces R61 enzyme β-lactam antibiotic complexes were compared with those of the complexes formed between the same antibiotics and either the membrane-bound transpeptidase from Streptomyces R61 or the exocellular dd-carboxypeptidase-transpeptidase of Streptomyces R39. [less ▲]

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See detailMolecular weight, amino acid composition and physicochemical properties of the exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R39
Frère, Jean-Marie ULg; Moreno, Ramon; Ghuysen, Jean-Marie ULg

in Biochemical Journal (1974), 143(1), 233-240

The exocellular dd-carboxypeptidase-transpeptidase from Streptomyces R39 was purified to protein homogeneity and in milligram amounts. The isolated enzyme consisted of one polypeptide chain of molecular ... [more ▼]

The exocellular dd-carboxypeptidase-transpeptidase from Streptomyces R39 was purified to protein homogeneity and in milligram amounts. The isolated enzyme consisted of one polypeptide chain of molecular weight about 53300. Its amino acid composition and several physicochemical properties were determined and compared with those of the exo-cellular dd-carboxypeptidase-transpeptidase from Streptomyces R61. [less ▲]

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See detailOccurrence of D-alanyl-(D)-meso-diaminopimelic acid and meso-diaminopimelyl-meso-diaminopimelic acid interpeptide linkages in the peptidoglycan of Mycobacteria
Wietzerbin, Juana; Das, Bhupsh C.; Petit, Jean-François et al

in Biochemistry (1974), 13(17), 3471-3476

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See detailMembrane-bound transpeptidase and penicillin binding sites in Streptomyces strain R61
Marquet, Alberto; Dusart, Jean; Ghuysen, Jean-Marie ULg et al

in European Journal of Biochemistry (1974), 46(3), 515-523

High-affinity penicillin binding sites from which the antibiotic could not be removed by washings at 4°C in 0.017 M K2HPO4 or 0.05 M Tris-HCl pH 7.5, were shown to occur in the isolated membranes of ... [more ▼]

High-affinity penicillin binding sites from which the antibiotic could not be removed by washings at 4°C in 0.017 M K2HPO4 or 0.05 M Tris-HCl pH 7.5, were shown to occur in the isolated membranes of Streptomyces R61. These sites caused the attachment of 25 picomoles of [14C]benzylpenicillin per milligram membrane protein. Penicillins and cephalosporins competed for the same binding sites. The antibiotic concentrations which excluded [14C]benzylpenicillin from 50% of the binding sites were those which inhibited by 50% the membrane-bound transpeptidase. The same rate constant (about 1 × 10−4 s−1) for the dissociation of the benzylpenicillin membrane complex at 37°C and in 0.017 M K2HPO4, was calculated either from the release of the radioactivity (using [14C]benzylpenicillin) or from the recovery of the transpeptidase activity. These observations supported the conclusion that the high-affinity binding sites in the isolated membranes were the transpeptidase molecules. All the complexes formed between the membranes and the various penicillins and cephalosporins examined were reversible at 37°C and in 0.017 M K2HPO4 at least with regard to the transpeptidase. Depending upon the antibiotics, the rate constants for the dissociation of these complexes varied from 3.3 × 10−3 to 0.73 × 10−4 s−1. The radioactivity released through the dissociation of [14C]benzylpenicillin membrane complex occurred mainly in the form of a compound which behaved as [W]-benzylpenicilloic acid both by paper electrophoresis and thin-layer chromatography. It was impossible to choose between several possible mechanisms for the release of the antibiotic molecule. [less ▲]

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See detailMembrane-bound DD-carboxypeptidase and LD-transpeptidase of Streptococcus faecalis ATCC 9790
Coyette, Jacques; Perkins, Harnold R.; Polacheck, Itzhack et al

in European Journal of Biochemistry (1974), 44(2), 459-468

Isolated membranes of Streptococcus faecalis ATCC 9790 exhibit DD-carboxypeptidase activity (standard reaction: Ac2-l-Lys-d-Ala-d-Ala →d-alanine + Ac2-l-Lys-d-Ala) and ld-trans-peptidase activity ... [more ▼]

Isolated membranes of Streptococcus faecalis ATCC 9790 exhibit DD-carboxypeptidase activity (standard reaction: Ac2-l-Lys-d-Ala-d-Ala →d-alanine + Ac2-l-Lys-d-Ala) and ld-trans-peptidase activity (standard reaction: Ac2-l-Lys-d-Ala + acceptor →d-alanine + Ac2-l-Lys-acceptor). The DD-carboxypeptidase activity has a considerable specificity for peptides with a C-terminal l-R3-d-Ala-d-Ala sequence where R3 is an amino acid residue and a long side-chain at the l-R3 position. A corresponding DD-transpeptidation reaction yielding the product Ac2-l-Lys-d-Ala-d-[14C]Ala from the system Ac2-l-Lys-d-Ala-d-Ala-f-d-[14C] alanine was not detected. The ld-transpeptidase activity has a considerable specificity for peptide donors that have an Nα-substituted, C-terminal l-R3-d-Ala sequence with a free ω-amino group at the end of a long side-chain at the l-R3 position, and a considerable specificity for amino group acceptors that are located on a d-carbon in α-position to a free carboxyl group. In the absence of acceptor, hydrolysis of the dipeptide Ac2-l-Lys-d-Ala (ld-carboxypeptidase activity) was not observed. Both DD-carboxypeptidase and ld-transpeptidase activities are inhibited by β-lactam antibiotics, but their relative sensitivity differs according to the particular antibiotic used. [less ▲]

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See detailThe DD-carboxypeptidase-transpeptidase system in Escherichia coli mutant strain
Pollock, J. J.; Nguyen-Distèche, Martine ULg; Ghuysen, Jean-Marie ULg et al

in Annals of the New York Academy of Sciences (1974), 235

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See detailThe penicillin receptor in Streptomyces
Ghuysen, Jean-Marie ULg; Leyh-Bouille, M.; Frère, Jean-Marie ULg et al

in Annals of the New York Academy of Sciences (1974), 235

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See detailEnzymes involved in wall peptide crosslinking in Escherichia coli K12, strain 44
Nguyen-Distèche, Martine ULg; Ghuysen, Jean-Marie ULg; Pollock, Jerry J. et al

in European Journal of Biochemistry (1974), 41(3), 447-455

By using the glutamate-amidated tetrapeptide l-alanyl-d-isoglutaminyl-(l)-meso-diamino-pimelyl-(l)-d-alanine as a probe, there appears to exist in the membranes of Escherichia coli K12 strain 44 a dd ... [more ▼]

By using the glutamate-amidated tetrapeptide l-alanyl-d-isoglutaminyl-(l)-meso-diamino-pimelyl-(l)-d-alanine as a probe, there appears to exist in the membranes of Escherichia coli K12 strain 44 a dd-carboxypoptidase-transpeptidase system which does not recognize this peptide and a dd-carboxypoptidase-transpeptidase system which recognizes it. The dd-carboxypeptidase-endopeptidase system is essentially hydrolytic. It catalyzes the hydrolysis of UDP-N-acetyl-muramyl-pentapeptide into UDP-N-acetylmuramyl-tetrapeptide and the hydrolysis of the wall peptidoglycan peptide dimer into monomers. These activities are not inhibited by the glutamate-amidated tetrapeptide. The system may consist either of two enzyme proteins having predominantly carboxypeptidase activity and endopeptidase activity, respectively, or of one enzyme protein of which the functioning would depend upon the environmental conditions. The dd-carboxypeptidase-transpeptidase system (a) catalyzes concomitant hydrolysis (carboxypeptidase activity) and transfer (natural model transpeptidase activity) reactions with the pentapeptide l-alanyl-γ-d-glutamyl-(l)-meso-diaminopimelyl-(l)-d-alanyl-d-alanine. The transfer reaction leads to the synthesis of a dimer that is identical to the one which occurs in the E. coli wall peptidoglycan; (b) utilizes the glutamate-amidated tetrapeptide as an acceptor. Simultaneous exposure of the pentapeptide and the glutamate-amidated tetrapeptide to the enzyme system leads to the formation of an hybrid monoamidated peptide dimer and causes a decreased hydrolysis of the pentapeptide; (c) by virtue of its own carboxypeptidase activity, it appears to exert some endopeptidase activity. Both carboxypeptidase and endopeptidase activities of this system are inhibited by the glutamate-amidated tetrapeptide, but this represents only a small fraction of the total hydrolytic activity of the membrane Brij-36T extract. (d) The system catalyzes an unnatural model transpeptidation reaction in which glycine replaces d-alanine at the C-terminal position of the nucleotide UDP-N-acetylmuramyl-pentapeptide. This system may also consist either of two enzyme proteins having predominantly natural model transpeptidase activity and unnatural model transpeptidase activity, respectively, or of one enzyme protein of which the functioning would depend upon the environmental conditions. Whatever the exact situation, the E. colidd-carboxypeptidase-transpeptidase system is in many respects, similar to the dd-carboxy-peptidase-transpeptidase single polypeptide enzymes isolated from Streptomyces strains R39 and R61. [less ▲]

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See detailSensitivity to ampicillin and cephalothin of enzymes involved in wall peptide crosslinking in Escherichia coli K12, strain 44
Nguyen-Distèche, Martine ULg; Pollock, Jerry J.; Ghuysen, Jean-Marie ULg et al

in European Journal of Biochemistry (1974), 41(3), 457-463

After extraction of the membranes of Escherichia coli K12 strain 44 by Brij-36T, each of the four enzyme activities (natural transpeptidase, unnatural transpeptidase, carboxypeptidase and endopeptidase ... [more ▼]

After extraction of the membranes of Escherichia coli K12 strain 44 by Brij-36T, each of the four enzyme activities (natural transpeptidase, unnatural transpeptidase, carboxypeptidase and endopeptidase) of the wall peptide crosslinking system, occurs in two forms characterized by large differences in their sensitivity to ampicillin (but much smaller differences in their sensitivity to cephalothin). The fractionation of the enzyme activities into two groups of low and high sensitivity to ampicillin is achieved essentially by chromatography of the membrane extract on DEAE-cellulose. [less ▲]

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See detailComment la Pénicilline tue les bactéries
Ghuysen, Jean-Marie ULg; Leyh-Bouille, Mélina; Frère, Jean-Marie ULg et al

in Annales de Microbiologie (1974), 125 B(2), 209-210

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See detailThe penicillin Target in Bacteria
Ghuysen, Jean-Marie ULg; Leyh-Bouille, Mélina; Frère, Jean-Marie ULg et al

in Spencer, B. (Ed.) Industrial Aspects of Biochemistry (FEBS Proceedings) (1974)

The bacterial target of beta-lactam antibiotics consists of a set of multiple, membrane-bound receptors. Some of them have been characterized as DD-carboxypeptidases. The DD-carboxypeptidases catalyse the ... [more ▼]

The bacterial target of beta-lactam antibiotics consists of a set of multiple, membrane-bound receptors. Some of them have been characterized as DD-carboxypeptidases. The DD-carboxypeptidases catalyse the opening of amide bonds and transfer the carbonyl carbon to an exogenous nucleophile, and are specifically designed to operate on the D-Ala-D-Ala linkageof L-R-D-Ala-D-Ala terminated peptides (where R is most often a diamino acid residue). The R61, R39 and several Bacilli DD-carboxypeptidases are known to be serine-enzymes and the G DD-carboxypeptidase has been characterized as a metallo (Zn ions) enzyme. Both the R61 and the G enzymes have been crystallized. In turn, the S. faecalis 43,000-Mr DD-carboxypeptidase, which is inhibited by low dose levels of pCMB, might be a thiol-enzyme. The goal pursued is the understanding of the mechanistic properties and functioning of the active centers of the DD-carboxypeptidases at the molecular and atomic levels. The research program involves 1) further characterization of the S. faecalis enzyme (which can be obtained in a water-soluble form); 2) isolation of various Streptomyces membrane-bound enzymes in a truly water-soluble form; 3) sequencing of the G and R61 enzymes; 4) the 2.8 A structure analysis of the G enzyme. (A similar study is conducted by Dr. J.R. Knox at the University of Connecticut, on the R61 enzyme, which enzyme is prepared and purified in this laboratory and then sent to Storrs); 5) conformational studies and quantitative structure activity relationships (QSAR). Source du résumé : http://www.researchcrossroads.org/index.php?view=article&id=50%3Agrant-details&option=com_content&Itemid=64&grant_id=4296130 [less ▲]

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See detailFractionation of the DD-carboxypeptidase-transpeptidase activities solubilized from membranes of Escherichia coli K12, strain 44
Pollock, Jerry J.; Nguyen-Disteche, Martine; Ghuysen, Jean-Marie ULg et al

in European Journal of Biochemistry (1974), 41(3), 439-446

A transpeptidase activity in Escherichia coli was measured independently of other enzymes involved in peptidoglycan synthesis by quantitating the formation of UDP-N-acetylmuramyl-l-alanyl-y-d-glutamyl-(l ... [more ▼]

A transpeptidase activity in Escherichia coli was measured independently of other enzymes involved in peptidoglycan synthesis by quantitating the formation of UDP-N-acetylmuramyl-l-alanyl-y-d-glutamyl-(l)-meso-diaminopimelyl-(l)-d-alanyl-[14C]glycine when UDP-N-acetylmuramyl-l-alanyl-y-d-glutamyl-(l)-meso-diaminopimelyl-(l-d-alanyl-d-alanine was used as donor substrate and [14C]glycine as acceptor in a transfer reaction. After extraction of membrane envelopes with Brij-36T and subsequent ammonium sulfate precipitation, DEAE-cellulose chromatography revealed two major fractions; one not adsorbed to the ion-exchange resin and the other adsorbed. The fraction which was bound to DEAE-cellulose was bound to and could be eluted from an ampicillin affinity chromatography system while the fraction not bound to DEAE-cellulose was also not bound to the ampicillin column. Both unbound and bound ampicillin fractions exhibited dd-carboxypeptidase and transpeptidase activities although for equivalent dd-carboxypeptidase activity, the bound ampicillin fraction required about five times more glycine acceptor to achieve the same amount of transpeptidation as the unbound ampicillin fraction. [less ▲]

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See detailBinding of beta-lactam antibiotics to the exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R39
Frère, Jean-Marie ULg; Ghuysen, Jean-Marie ULg; Reynolds, Peter E. et al

in Biochemical Journal (1974), 143(1), 241-249

Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase-transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic-enzyme complexes. At saturation, the ... [more ▼]

Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase-transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic-enzyme complexes. At saturation, the molar ratio of chromogenic cephalosporin 87-312 to enzyme was 1.3:1, but this discrepancy might be due to a lack of accuracy in the measurement of the antibiotic. Spectrophotometric studies showed that binding of cephaloridine and cephalosporin 87-312 to the enzyme caused opening of their beta-lactam rings. Benzylpenicillin and cephalosporin 87-312 competed for the same site on the free enzyme, suggesting that binding of benzylpenicillin also resulted in the opening of its beta-lactam ring. In Tris-NaCl-MgCl(2) buffer at pH7.7 and 37 degrees C, the rate constants for the dissociation of the antibiotic-enzyme complexes were 2.8x10(-6), 1.5x10(-6) and 0.63x10(-6)s(-1) (half-lives 70, 130 and 300h) for benzylpenicillin, cephalosporin 87-312 and cephaloridine respectively. During the process, the protein underwent reactivation. The enzyme that was regenerated from its complex with benzylpenicillin was as sensitive to fresh benzylpenicillin as the native enzyme. With [(14)C]benzylpenicillin, the released radioactive compound was neither benzylpenicillin nor benzylpenicilloic acid. The Streptomyces R39 enzyme thus behaved as a beta-lactam-antibiotic-destroying enzyme but did not function as a beta-lactamase. Incubation at 37 degrees C in 0.01m-phosphate buffer, pH7.0, and in the same buffer supplemented with sodium dodecyl sulphate caused a more rapid reversion of the [(14)C]benzylpenicillin-enzyme complex. The rate constants were 1.6x10(-5)s(-1) and 0.8x10(-4)s(-1) respectively. Under these conditions, however, there was no concomitant reactivation of the enzyme and the released radioactive compound(s) appeared not to be the same as before. The Streptomyces R39 enzyme and the exocellular dd-carboxypeptidase-transpeptidase from Streptomyces R61 appeared to differ from each other with regard to the topography of their penicillin-binding site. [less ▲]

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