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W Kurzatkowski - One of the best experts on this subject based on the ideXlab platform.

  • Affinity of exocellular DD-Carboxypeptidase/Transpeptidase from Saccharopolyspora erythraea PZH TZ-575 to beta-lactam compounds
    Medycyna doswiadczalna i mikrobiologia, 1999
    Co-Authors: J Solecka, W Kurzatkowski
    Abstract:

    The DD-Carboxypeptidase/Transpeptidases (DD-peptidases) involved in bacterial cell wall metabolism, catalyse the attack of C-terminal D-alanyl-D-alanine peptide bond of the peptydoglycan precursor. These enzymes are inactivated by beta-lactam antibiotics. DD-peptidase from Saccharopolyspora erythraea PZH TZ 64-575 was purified by the use of DEAE-cellulose, Sephadex G-100, Q-Sepharose resins and FPLC (Mono Q). After each step the effluent was concentrated by Amicon ultrafiltration. The purified enzyme showed DD-Carboxypeptidase specific activity of 50.9 U/mg. The enzyme exhibited high affinity to beta-lactam compounds e.g. cefamandole, cefapirin, cefradin 1.5-2.6 x 10(-8) M. It was used to screen strains from the Culture Collection of the National Institute of Hygiene in Warsaw for the production of DD-peptidase inhibitors.

  • affinity of exocellular dd Carboxypeptidase Transpeptidase from saccharopolyspora erythraea pzh tz 575 to beta lactam compounds
    Medycyna doświadczalna i mikrobiologia, 1999
    Co-Authors: J Solecka, W Kurzatkowski
    Abstract:

    The DD-Carboxypeptidase/Transpeptidases (DD-peptidases) involved in bacterial cell wall metabolism, catalyse the attack of C-terminal D-alanyl-D-alanine peptide bond of the peptydoglycan precursor. These enzymes are inactivated by beta-lactam antibiotics. DD-peptidase from Saccharopolyspora erythraea PZH TZ 64-575 was purified by the use of DEAE-cellulose, Sephadex G-100, Q-Sepharose resins and FPLC (Mono Q). After each step the effluent was concentrated by Amicon ultrafiltration. The purified enzyme showed DD-Carboxypeptidase specific activity of 50.9 U/mg. The enzyme exhibited high affinity to beta-lactam compounds e.g. cefamandole, cefapirin, cefradin 1.5-2.6 x 10(-8) M. It was used to screen strains from the Culture Collection of the National Institute of Hygiene in Warsaw for the production of DD-peptidase inhibitors.

Jean-marie Frère - One of the best experts on this subject based on the ideXlab platform.

  • Specificity inversion of Ochrobactrum anthropi D-aminopeptidase to a D,D-Carboxypeptidase with new penicillin binding activity by directed mutagenesis.
    Protein Science, 2005
    Co-Authors: Michaël Delmarcelle, Jean-marie Frère, Marie-caroline Boursoit, Patrice Filée, Stéphane Baurin, Bernard Joris
    Abstract:

    The serine penicillin-recognizing proteins have been extensively studied. They show a wide range of substrate specificities accompanied by multidomain features. Their adaptation capacity has resulted in the emergence of pathogenic bacteria resistant to beta-lactam antibiotics. The most divergent enzymatic activities in this protein family are those of the Ochrobactrum anthropi D-aminopeptidase and of the Streptomyces R61 D,D-Carboxypeptidase/Transpeptidase. With the help of structural data, we have attempted to identify the factors responsible for this opposite specificity. A loop deletion mutant of the Ochrobactrum anthropi D-aminopeptidase lost its original activity in favor of a new penicillin-binding activity. D-aminopeptidase activity of the deletion mutant can be restored by complementation with another deletion mutant corresponding to the noncatalytic domain of the wild-type enzyme. By a second step site-directed mutagenesis, the specificity of the Ochrobactrum anthropi D-aminopeptidase was inverted to a D,D-Carboxypeptidase specificity. These results imply a core enzyme with high diversity potential surrounded by specificity modulators. It is the first example of drastic specificity change in the serine penicillin-recognizing proteins. These results open new perspectives in the conception of new enzymes with nonnatural specificities. The structure/specificity relationship in the serine penicillin-recognizing proteins are discussed.

  • Molecular evolution of bacterial β-lactam resistance
    Chemistry & Biology, 1996
    Co-Authors: James R. Knox, Paul C. Moews, Jean-marie Frère
    Abstract:

    Abstract Background: Two groups of penicillin-destroying enzymes, the class A and class C β-lactamases, may have evolved from bacterial Transpeptidases that transfer x-d-Ala-d-Ala peptides to the growing peptidoglycan during cell wall synthesis. Both the Transpeptidases and the β-lactamases are acylated by β-lactam antibiotics such as penicillin, which mimic the peptide, but breakdown and removal of the antibiotic is much faster in the β-lactamases, which lack the ability to process d-Ala-d-Ala peptides. Stereochemical factors driving this evolution in specificity are examined. Results: We have compared the crystal structures of two classes of β-lactamases and a β-lactam-sensitive d-alanyl-d-alanine-Carboxypeptidase/Transpeptidase (DD-peptidase). The class C β-lactamase is more similar to the DD-peptidase than to another β-lactamase of class A. Conclusions: The two classes of β-lactamases appear to have developed from an ancestral protein along separate evolutionary paths. Structural differentiation of the β-lactamases from the DD-peptidases appears to follow differences in substrate shapes. The structure of the class A β-lactamase has been further optimized to exclude d-alanyl peptides and process penicillin substrates with near catalytic perfection. Keywords: drug resistance, enzymology, penicillin antibiotics, protein ancestry Received: 7 October 1996

  • Point mutations of two arginine residues in the Streptomyces R61 DD-peptidase.
    Biochemical Journal, 1992
    Co-Authors: C Bourguignon-bellefroid, Jean-marie Ghuysen, Bernard Joris, J. Van Beeumen, Jean-marie Frère
    Abstract:

    Incubation of the exocellular DD-Carboxypeptidase/Transpeptidase of Streptomyces R61 with phenylglyoxal resulted in a time-dependent decrease in the enzyme activity. This inactivation was demonstrated to be due to modification of the Arg-99 side chain. In consequence, the role of that residue was investigated by site-directed mutagenesis. Mutation of Arg-99 into leucine appeared to be highly detrimental to enzyme stability, reflecting a determining structural role for this residue. The conserved Arg-103 residue was also substituted by using site-directed mutagenesis. The modification to a serine residue yielded a stable enzyme, the catalytic properties of which were similar to those of the wild-type enzyme. Thus Arg-103, although strictly conserved or replaced by a lysine residue in most of the active-site penicillin-recognizing proteins, did not appear to fulfil any essential role in either the enzyme activity or structure.

J Solecka - One of the best experts on this subject based on the ideXlab platform.

  • Affinity of exocellular DD-Carboxypeptidase/Transpeptidase from Saccharopolyspora erythraea PZH TZ-575 to beta-lactam compounds
    Medycyna doswiadczalna i mikrobiologia, 1999
    Co-Authors: J Solecka, W Kurzatkowski
    Abstract:

    The DD-Carboxypeptidase/Transpeptidases (DD-peptidases) involved in bacterial cell wall metabolism, catalyse the attack of C-terminal D-alanyl-D-alanine peptide bond of the peptydoglycan precursor. These enzymes are inactivated by beta-lactam antibiotics. DD-peptidase from Saccharopolyspora erythraea PZH TZ 64-575 was purified by the use of DEAE-cellulose, Sephadex G-100, Q-Sepharose resins and FPLC (Mono Q). After each step the effluent was concentrated by Amicon ultrafiltration. The purified enzyme showed DD-Carboxypeptidase specific activity of 50.9 U/mg. The enzyme exhibited high affinity to beta-lactam compounds e.g. cefamandole, cefapirin, cefradin 1.5-2.6 x 10(-8) M. It was used to screen strains from the Culture Collection of the National Institute of Hygiene in Warsaw for the production of DD-peptidase inhibitors.

  • affinity of exocellular dd Carboxypeptidase Transpeptidase from saccharopolyspora erythraea pzh tz 575 to beta lactam compounds
    Medycyna doświadczalna i mikrobiologia, 1999
    Co-Authors: J Solecka, W Kurzatkowski
    Abstract:

    The DD-Carboxypeptidase/Transpeptidases (DD-peptidases) involved in bacterial cell wall metabolism, catalyse the attack of C-terminal D-alanyl-D-alanine peptide bond of the peptydoglycan precursor. These enzymes are inactivated by beta-lactam antibiotics. DD-peptidase from Saccharopolyspora erythraea PZH TZ 64-575 was purified by the use of DEAE-cellulose, Sephadex G-100, Q-Sepharose resins and FPLC (Mono Q). After each step the effluent was concentrated by Amicon ultrafiltration. The purified enzyme showed DD-Carboxypeptidase specific activity of 50.9 U/mg. The enzyme exhibited high affinity to beta-lactam compounds e.g. cefamandole, cefapirin, cefradin 1.5-2.6 x 10(-8) M. It was used to screen strains from the Culture Collection of the National Institute of Hygiene in Warsaw for the production of DD-peptidase inhibitors.

Frère Jean-marie - One of the best experts on this subject based on the ideXlab platform.

  • Point Mutations of Two Arginine Residues in the Streptomyces R61 Dd-Peptidase
    'Portland Press Ltd.', 1992
    Co-Authors: Bourguignon-bellefroid Catherine, Joris Bernard, Ghuysen Jean-marie, Van Beeumen Jacques, Frère Jean-marie
    Abstract:

    peer reviewedaudience: researcher, professionalIncubation of the exocellular DD-Carboxypeptidase/Transpeptidase of Streptomyces R61 with phenylglyoxal resulted in a time-dependent decrease in the enzyme activity. This inactivation was demonstrated to be due to modification of the Arg-99 side chain. In consequence, the role of that residue was investigated by site-directed mutagenesis. Mutation of Arg-99 into leucine appeared to be highly detrimental to enzyme stability, reflecting a determining structural role for this residue. The conserved Arg-103 residue was also substituted by using site-directed mutagenesis. The modification to a serine residue yielded a stable enzyme, the catalytic properties of which were similar to those of the wild-type enzyme. Thus Arg-103, although strictly conserved or replaced by a lysine residue in most of the active-site penicillin-recognizing proteins, did not appear to fulfil any essential role in either the enzyme activity or structure

  • Point Mutations of Two Arginine Residues in the Streptomyces R61 Dd-Peptidase
    1992
    Co-Authors: Bourguignon-bellefroid Catherine, Joris Bernard, Ghuysen Jean-marie, Van Beeumen Jacques, Frère Jean-marie
    Abstract:

    Incubation of the exocellular DD-Carboxypeptidase/Transpeptidase of Streptomyces R61 with phenylglyoxal resulted in a time-dependent decrease in the enzyme activity. This inactivation was demonstrated to be due to modification of the Arg-99 side chain. In consequence, the role of that residue was investigated by site-directed mutagenesis. Mutation of Arg-99 into leucine appeared to be highly detrimental to enzyme stability, reflecting a determining structural role for this residue. The conserved Arg-103 residue was also substituted by using site-directed mutagenesis. The modification to a serine residue yielded a stable enzyme, the catalytic properties of which were similar to those of the wild-type enzyme. Thus Arg-103, although strictly conserved or replaced by a lysine residue in most of the active-site penicillin-recognizing proteins, did not appear to fulfil any essential role in either the enzyme activity or structure.Peer reviewe

Bourguignon-bellefroid Catherine - One of the best experts on this subject based on the ideXlab platform.

  • Point Mutations of Two Arginine Residues in the Streptomyces R61 Dd-Peptidase
    'Portland Press Ltd.', 1992
    Co-Authors: Bourguignon-bellefroid Catherine, Joris Bernard, Ghuysen Jean-marie, Van Beeumen Jacques, Frère Jean-marie
    Abstract:

    peer reviewedaudience: researcher, professionalIncubation of the exocellular DD-Carboxypeptidase/Transpeptidase of Streptomyces R61 with phenylglyoxal resulted in a time-dependent decrease in the enzyme activity. This inactivation was demonstrated to be due to modification of the Arg-99 side chain. In consequence, the role of that residue was investigated by site-directed mutagenesis. Mutation of Arg-99 into leucine appeared to be highly detrimental to enzyme stability, reflecting a determining structural role for this residue. The conserved Arg-103 residue was also substituted by using site-directed mutagenesis. The modification to a serine residue yielded a stable enzyme, the catalytic properties of which were similar to those of the wild-type enzyme. Thus Arg-103, although strictly conserved or replaced by a lysine residue in most of the active-site penicillin-recognizing proteins, did not appear to fulfil any essential role in either the enzyme activity or structure

  • Point Mutations of Two Arginine Residues in the Streptomyces R61 Dd-Peptidase
    1992
    Co-Authors: Bourguignon-bellefroid Catherine, Joris Bernard, Ghuysen Jean-marie, Van Beeumen Jacques, Frère Jean-marie
    Abstract:

    Incubation of the exocellular DD-Carboxypeptidase/Transpeptidase of Streptomyces R61 with phenylglyoxal resulted in a time-dependent decrease in the enzyme activity. This inactivation was demonstrated to be due to modification of the Arg-99 side chain. In consequence, the role of that residue was investigated by site-directed mutagenesis. Mutation of Arg-99 into leucine appeared to be highly detrimental to enzyme stability, reflecting a determining structural role for this residue. The conserved Arg-103 residue was also substituted by using site-directed mutagenesis. The modification to a serine residue yielded a stable enzyme, the catalytic properties of which were similar to those of the wild-type enzyme. Thus Arg-103, although strictly conserved or replaced by a lysine residue in most of the active-site penicillin-recognizing proteins, did not appear to fulfil any essential role in either the enzyme activity or structure.Peer reviewe