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See detailSite-Directed Mutagenesis of the Actinomadura R39 DD-Peptidase
Zhao, GuoHua; Duez, Colette ULg; Forceille, Christine et al

in Biochemical Journal (1997), 327(2), 377-381

The role of various residues in the conserved structural elements of the Actinomadura R39 penicillin-sensitive dd-peptidase has been studied by site-directed mutagenesis. Replacement of Ser-298 of the ... [more ▼]

The role of various residues in the conserved structural elements of the Actinomadura R39 penicillin-sensitive dd-peptidase has been studied by site-directed mutagenesis. Replacement of Ser-298 of the 'SDN loop' by Ala or Gly significantly decreased the kcat/Km value for the peptide substrate, but only by a factor of 15 and had little effect on the other catalytic properties. Mutations of Asn-300 of the same loop and of Lys-410 of the KTG triad yielded very unstable proteins. However, the N300S mutant could be purified as a fusion protein with thioredoxin that exhibited decreased rates of acylation by the peptide substrate and various cephalosporins. Similar fusion proteins obtained with the N300A, K410H and K410N mutants were unstable and their catalytic and penicillin-binding properties were very strongly affected. In transpeptidation reactions, the presence of the acceptor influenced the kcat/Km values, which suggested a catalytic pathway more complex than a simple partition of the acyl-enzyme between hydrolysis and aminolysis. These results are compared with those obtained with two other penicillin-sensitive enzymes, the Streptomyces R61 dd-peptidase and Escherichia coli penicillin-binding protein (PBP) 5. [less ▲]

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See detailKinetic Study of Interaction between Brl 42715, Beta-Lactamases, and D-Alanyl-D-Alanine Peptidases
Matagne, André ULg; Ledent, Philippe; Monnaie, Didier et al

in Antimicrobial Agents and Chemotherapy (1995), 39(1), 227-31

A detailed kinetic study of the interactions between BRL 42715, a beta-lactamase-inhibiting penem, and various beta-lactamases (EC 3.5.2.6) and D-alanyl-D-alanine peptidases (DD-peptidases, EC 3.4.16.4 ... [more ▼]

A detailed kinetic study of the interactions between BRL 42715, a beta-lactamase-inhibiting penem, and various beta-lactamases (EC 3.5.2.6) and D-alanyl-D-alanine peptidases (DD-peptidases, EC 3.4.16.4) is presented. The compound was a very efficient inactivator of all active-site serine beta-lactamases but was hydrolyzed by the class B, Zn(2+)-containing enzymes, with very different kcat values. Inactivation of the Streptomyces sp. strain R61 extracellular DD-peptidase was not observed, and the Actinomadura sp. strain R39 DD-peptidase exhibited a low level of sensitivity to the compound. [less ▲]

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See detailSite-Directed Mutagenesis of the Streptomyces R61 Dd-Peptidase. Catalytic Function of the Conserved Residues around the Active Site and a Comparison with Class-a and Class-C Beta-Lactamases
Hadonou, Ayaovi Medard; Wilkin, Jean-Marc; Varetto, Louis ULg et al

in European Journal of Biochemistry (1992), 207(1), 97-102

The importance of various residues in the Streptomyces R61 penicillin-sensitive DD-peptidase has been assessed by site-directed mutagenesis. The replacement of the active Ser62 by a Cys residue yielded an ... [more ▼]

The importance of various residues in the Streptomyces R61 penicillin-sensitive DD-peptidase has been assessed by site-directed mutagenesis. The replacement of the active Ser62 by a Cys residue yielded an inactive protein which was also unable to recognize penicillin. The activity of the Lys65 → Arg mutant with the peptide and thiol ester substrates was decreased 100-200-fold and the rate of penicillin inactivation was decreased 20 000-fold or more. The mutant thus behaved as a poor, but penicillin-resistant, DD-peptidase. The other studied mutations, the mutations Phe358 → Leu, Tyr90 → Asn, Thr101 → Asn, Phe164 → Ala, Asp225 → Glu and Asp225 → Asn had little influence on the catalytic and penicillin-binding properties. The Asp225 mutants did not exhibit an increased sensitivity to cefotaxime. The Phe164 → Ala mutant was significantly more unstable than the wild-type enzyme. [less ▲]

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See detailThe active sites of the β-lactamases of Streptomyces cacaoi and Streptomyces albus G
Demeester, Fabien; Joris, Bernard ULg; Lenzini, Mauro V. et al

in Biochemical Journal (1987), 244(2), 427-432

The active-site serine of the extracellular beta-lactamases of Streptomyces cacaoi and Streptomyces albus G has been labelled with beta-iodopenicillanate. The determination of the sequence of the labelled ... [more ▼]

The active-site serine of the extracellular beta-lactamases of Streptomyces cacaoi and Streptomyces albus G has been labelled with beta-iodopenicillanate. The determination of the sequence of the labelled peptides obtained after trypsin digestion of the denatured proteins indicate both enzymes to be class A beta-lactamases. Surprisingly the two Streptomyces enzymes do not appear to be especially homologous, and none of them exhibited a high degree of homology with the Streptomyces R61 DD-peptidase. Our data confirm that, as a family of homologous enzymes, class A is rather heterogeneous, with only a small number of conserved residues in all members of the class. [less ▲]

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See detailDes-, syn- and anti-oxyimino-Δ3-cephalosporins. Intrinsic reactivity and reaction with RTEM-2 serine β-lactamase and D-alanyl-D-alanine-cleaving serine and zinc-containing peptidases
Laurent, Guy; Durant, François; Frère, Jean-Marie ULg et al

in Biochemical Journal (1984), 218(3), 933-937

The presence and configuration (syn or anti) of an oxyimino group in the 7 (beta)-acyl side chain of 3-cephems do not modify the intrinsic reactivity of the beta-lactam ring, but have highly enzyme ... [more ▼]

The presence and configuration (syn or anti) of an oxyimino group in the 7 (beta)-acyl side chain of 3-cephems do not modify the intrinsic reactivity of the beta-lactam ring, but have highly enzyme-specific effects. When compared with the corresponding desoxyimino beta-lactam compound: (i) with the plasmid-mediated Escherichia coli RTEM-2 serine beta-lactamase, the substrate activity of the anti isomer is increased and that of the syn isomer is decreased; (ii) with the Streptomyces R61 serine D-alanyl-D-alanine cleaving peptidase (a highly penicillin-sensitive enzyme), the rate of enzyme acylation is not or only little affected when the oxyimino group is in the syn configuration, but is decreased when the oxyimino group is in the anti configuration; (iii) with the Actinomadura R39 serine D-alanyl-D-alanine-cleaving peptidase (an exceedingly highly penicillin-sensitive enzyme), the rate of enzyme acylation is unaffected whatever the configuration of the substituent. The oxidation of the sulphur atom of the dihydrothiazine ring on the beta-face of the molecule makes it both a poorer inactivator of the DD-peptidases and a poorer substrate of the beta-lactamase. The Streptomyces albus G Zn2+-containing D-alanyl-D-alanine-cleaving peptidase (a highly penicillin-resistant enzyme) remains highly resistant to all compounds tested. [less ▲]

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See detailInteraction between monobactams and model D-alanyl-D-alanine-cleaving peptidases
Frère, Jean-Marie ULg; Klein, Daniel; Kelly, Judith A et al

in FEMS Microbiology Letters (1984), 21

Several monobactams reacted with the serine dd-peptidases of Streptomyces R61 and Actinomadura R39 in a manner similar to that of bicyclic penicillins and cephalosporins. The dissociation constants of the ... [more ▼]

Several monobactams reacted with the serine dd-peptidases of Streptomyces R61 and Actinomadura R39 in a manner similar to that of bicyclic penicillins and cephalosporins. The dissociation constants of the Michaelis complexes formed between the R61 enzyme and sulfazecin (32 μM) and between the R39 peptidase and SQ 26324 (0.35 μM) had the lowest values ever observed with any β-lactam compound, suggesting an excellent fit of these two monobactams with the active sites of the respective enzymes. Azthreonam had a very poor inactivating potency, confirming its high selective reactivity towards the penicillin binding protein No. 3 of Escherichia coli. The Zn2+dd-peptidase (from Streptomyces albus G) had a high intrinsic resistance to β-lactam compounds whether they possessed a mono- or a bicyclic structure. [less ▲]

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See detailΔ2- and Δ3-Cephalosporins, penicillinate, and 6-unsubstituted penems. Intrinsic reactivity and interaction with β-lactamases and D-alanyl-D-alanine-cleaving serine peptidases
Frère, Jean-Marie ULg; Kelly, Judith A; Klein, Daniel et al

in Biochemical Journal (1982), 203(1), 223-234

The intrinsic reactivity of delta 2- and delta 3-deacetoxy-7-phenylacetamidocephalosporanates, penicillanate, unsubstituted, 2-methyl- and 2-phenyl-penems and other beta-lactam antibiotics has been ... [more ▼]

The intrinsic reactivity of delta 2- and delta 3-deacetoxy-7-phenylacetamidocephalosporanates, penicillanate, unsubstituted, 2-methyl- and 2-phenyl-penems and other beta-lactam antibiotics has been expressed in terms of the second-order rate constant (M-1.s-1(OH-)) for the hydrolysis of the beta-lactam amide bond by OH- at 37 degrees C. The values thus obtained have been compared with the second-order rate constants (M-1.s-1(enzyme) for the opening of the same beta-lactam amide bond during interaction with the beta-lactamases of Streptomyces albus G and Actinomadura R39 and the D-alanyl-D-alanine-cleaving serine peptidases of Streptomyces R61 and Actinomadura R39. Depending on the cases, the accelerating effect due to enzyme action and expressed by the ratio M-1.s-1(enzyme)/M-1.s-1(OH) varies from less than 2 to more than 1 x 10(6). The primary parameter that governs enzyme action is the goodness of fit of the beta-lactam molecule to the enzyme cavity rather than its intrinsic reactivity. With the D-alanyl-D-alanine-cleaving serine peptidases, the three penems studied form intermediate complexes characterized by very short half lives of 14-100 s, values significantly lower than those exhibited by most beta-lactam compounds. [less ▲]

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See detailThe exocellular beta-lactamase of Streptomyces albus G.Purification, properties and comparison with the exocellular DD-carboxypeptidase
Duez, Colette ULg; Frère, Jean-Marie ULg; Klein, Daniel et al

in Biochemical Journal (1981), 193(1), 75-82

The exocellular beta-lactamase of Streptomyces albus G has been purified to near protein homogeneity. It consists of one single polypeptide chain of mol.wt. 30 000-31 000, has a rather low isoelectric ... [more ▼]

The exocellular beta-lactamase of Streptomyces albus G has been purified to near protein homogeneity. It consists of one single polypeptide chain of mol.wt. 30 000-31 000, has a rather low isoelectric point (at pH 6.0) and contains less lysine (2.1%) and more half-cystine residues than most beta-lactamases from other Gram-positive bacteria. Penicillins are much better substrates than delta 3-cephalosporins; the catalytic-centre activity of good penicillin substrates is 333-500 s-1. The exocellular, mol.wt. 17 000 DD-carboxypeptidase of S. albus G [previously purified to protein homogeneity; Duez, Frere, Geurts, Ghuysen, Dierickx & Delcambe (1978) Biochem. J. 175, 793-800] behaves as an exceedingly poor beta-lactamase, hydrolysing benzylpenicillin into benzylpenicilloate 5 x 10(-6)-fold less rapidly than does the exocellular beta-lactamase. To all appearances, the beta-lactamase has no bivalent cation requirement whereas, as shown elsewhere [Dideberg, Charlier, Dupont, Vermeire, Frere & Ghuysen (1980) FEBS Lett. 117, 212-214, and Dideberg, Joris, Frere, Ghuysen, Weber, Robaye, Delbrouck & Roelands (1980) FEBS Lett. 117, 215-218], the DD-carboxypeptidase possesses one essential Zn2+ ion per molecule. Peptide 'mapping' and immunological studies suggest that the two Streptomyces enzymes probably have very different structural and mechanistic properties. [less ▲]

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See detailEnzymatic method for rapid and sensitive determination of β-lactam antibiotics
Frère, Jean-Marie ULg; Klein, Daniel; Ghuysen, Jean-Marie ULg

in Antimicrobial Agents and Chemotherapy (1980), 18(4), 506-510

A rapid and sensitive procedure for the estimation of beta-lactam antibiotics is described which makes use of the ability of these antibiotics to inactivate the R39 DD-carboxypeptidase. Depending on the ... [more ▼]

A rapid and sensitive procedure for the estimation of beta-lactam antibiotics is described which makes use of the ability of these antibiotics to inactivate the R39 DD-carboxypeptidase. Depending on the values of the kinetic parameters which govern the reaction, the antibiotics fall into two groups. The lower limit for the quantitative estimation of the antibiotics of groups I and II is about 5 and 50 pmol/ml, respectively. The procedure has been adopted to biological fluids such as human sera and cows' milk. [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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