References of "Ghuysen, Jean-Marie"
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See detailInstrinct Resistance to beta-lactam antibiotics at the level of the enzyme sites. Many challenges, some achievements
Ghuysen, Jean-Marie ULg; Charlier, P.; Coyette, Jean et al

in Wiedemann, B.; Guysen, Jean-Marie; Spitzy, K. H. (Eds.) et al Symposium Mechanisms of resistance to beta-lactam antibiotics : Proceedings (1983)

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See detailThe active sites of the D-alanyl-D-alanine-cleaving peptidases
Charlier, Paulette ULg; Dideberg, Otto; Dive, Georges ULg et al

in Hakenbeck, Regine; Höltje, Joachim-Volker; Labischinski, Harald (Eds.) The Target Penicillin : the Murein Sacculus of Bacterial Cell Walls Architecture and Growth : Proceedings (1983)

The active site structures of D-alanyl-D-alanine-cleaving peptidases G, R61, R39, and K15 from Streptomyces and Actinomadura are discussed in relation to their substrate specificities and kinetic ... [more ▼]

The active site structures of D-alanyl-D-alanine-cleaving peptidases G, R61, R39, and K15 from Streptomyces and Actinomadura are discussed in relation to their substrate specificities and kinetic mechanisms. [less ▲]

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See detailFunction of Penicillin-binding protein 3 in Streptococcus Faecium
Coyette, Jacques; Somzé, Anne; Briquet, Jean-Jacques et al

in Hakenbeck, Regine; Höltje, Joachim-Volker; Labischinski, Harald (Eds.) The Target Penicillin : the Murein Sacculus of Bacterial Cell Walls Architecture and Growth : Proceedings (1983)

Cefotaxime at concns. around the min. inhibitory concn. (MIC, 5 μM) or below (0.1-1.0 μM) causes transformation of the normal S. faecium cells to bacilliform cells whose length increases with increasing ... [more ▼]

Cefotaxime at concns. around the min. inhibitory concn. (MIC, 5 μM) or below (0.1-1.0 μM) causes transformation of the normal S. faecium cells to bacilliform cells whose length increases with increasing duration of treatment. Septa are initiated but never reach completion. Affinity detn. for cefotaxime binding showed that the antibiotic binds preferentially to the 3 highest mol. wt. penicillin-binding proteins (PBP); PBP-2 and PBP-3 are about 100-fold more sensitive to cefotaxime than PBP-1. It appears, therefore, that PbP-2 and PBP-3 are involved in cell septation. This was confirmed by using cefatoxime in subinhibitory concns. (1 μM). From cell samples collected 30 and 60 min after addn. of cefotaxime, membranes were isolated, labeled with satg. [3H]benzylpenicillin and examd. by fluorog. Under these conditions only PBP-2 and PBP-3 were satd. by cefotaxime. At this stage no distinction could be made between the 2 proteins; either both or 1 of them may be involved in cell division. Cefoxitin produced morphol. alteration of a different nature than cefotaxime. The cefoxitin-treated cells had increased diam. and were slightly elongated. The most striking alteration was the frequent presence of conical poles contrasting with round poles obsd. in control cells. The morphol. alteration obsd. in cefoxitin-treated cells could be attributed to the inhibition of the function of PBP-1, PBP-2, or PBP-3. Elongated cells similar to those obtained with cefotaxime were not found with cefoxitin at concns. sufficient to sat. PBP-2. The main difference between cefotaxime- and cefoxitin-treated cells is that PBP-3 is satd. by cefotaxime but not altered at all by cefoxitin. Thus, septation inhibition must be due to the interaction of cefotaxime with PBP-3 [less ▲]

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See detailPenicillin-binding proteins and carboxypeptidase/transpeptidase activities in Proteus vulgaris P18 and its penicillin-induced stable L-forms
Rousset, André; Nguyen-Distèche, Martine ULg; Minck, Raymond et al

in Journal of Bacteriology (1982), 152(3), 1042-1048

The originally penicillin-induced, wall-less stable L-forms of Proteus vulgaris P18, isolated by Tulasne in 1949 and since then cultured in he absence of penicillin, have kept the ability to synthesize ... [more ▼]

The originally penicillin-induced, wall-less stable L-forms of Proteus vulgaris P18, isolated by Tulasne in 1949 and since then cultured in he absence of penicillin, have kept the ability to synthesize the seven penicillin-binding proteins and the various DD- and LD-peptidase activities found in the parental bacteria and known to be involved in wall peptidoglycan metabolism. The stable L-forms, however, secrete during growth both the highly penicillin-sensitive, DD-carboxy-peptidase-transpeptidase penicillin-binding protein PBP4 (which in normal bacteria is relatively loosely bound to the plasma membrane) and the penicillin-insensitive LD-carboxypeptidase (which in normal bacteria is located in the periplasmic region). [less ▲]

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See detailPenicillin target enzyme and the antibiotic binding site
Kelly, Judith A.; Moews, Paul C.; Knox, James R. et al

in Science (1982), 218(4571), 479-481

The three-dimensional structure of a penicillin-sensitive D-alanyl-carboxypeptidase-transpeptidase has been determined by x-ray crystallography to a resolution of 2.8 angstroms. The site of binding of the ... [more ▼]

The three-dimensional structure of a penicillin-sensitive D-alanyl-carboxypeptidase-transpeptidase has been determined by x-ray crystallography to a resolution of 2.8 angstroms. The site of binding of the beta-lactam antibiotics penicillin and cephalosporin has been located. These findings constitute direct observation of the interaction of beta-lactams with a transpeptidase enzyme and establish the feasibility of defining the molecular stereochemistry of this interaction for purposes of drug design. [less ▲]

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See detailIsolation of the membrane-bound 26 000-Mr penicillin-binding protein of Streptomyces strain K15 in the form of a penicillin-sensitive D-alanyl-D-alanine-cleaving transpeptidase
Nguyen-Distèche, Martine ULg; Leyh-Bouille, Mélina; Ghuysen, Jean-Marie ULg

in Biochemical Journal (1982), 207(1), 109-115

The membrane-bound, 26 000-Mr penicillin-binding protein of Streptomyces K15 has been isolated in the form of an effective, penicillin-sensitive D-alanyl-D-alanine-cleaving peptidase exhibiting high ... [more ▼]

The membrane-bound, 26 000-Mr penicillin-binding protein of Streptomyces K15 has been isolated in the form of an effective, penicillin-sensitive D-alanyl-D-alanine-cleaving peptidase exhibiting high transpeptidase activity (greater than 95%) and very low carboxy-peptidase activity (less than 5%). The penicillin-binding protein/transpeptidase can be extracted directly from the mycelium with N-cetyl-NNN-trimethylammonium bromide (Cetavlon) and subsequently obtained at 90% purity and with an 8000-fold specific enrichment (when compared with the activity of the isolated membranes) by a two-step procedure involving Sephadex filtration and affinity chromatography on ampicillin-linked CH Sepharose 4B in the presence of detergent. At saturating concentrations of the co-substrates diacetyl-L-Lys-D-Ala-D-Ala and Gly-Gly, the catalytic-centre activity is about 0.3 s-1. [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 detailPurification and properties of the exocellular β-lactamase of Actinomadura strain R39
Duez, Colette ULg; Frère, Jean-Marie ULg; Ghuysen, Jean-Marie ULg et al

in Biochimica et Biophysica Acta - General Subjects (1982), 700

The exocellular beta-lactamase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) of Actinomadura R39 consists of one single polypeptide chain of molecular weight about 15 200. It exhibits a highly ... [more ▼]

The exocellular beta-lactamase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) of Actinomadura R39 consists of one single polypeptide chain of molecular weight about 15 200. It exhibits a highly asymmetrical shape, has a low isoelectric point (at pH 5.0) and contains about 9.3% (w/w) of a polydeoxyribonucleotide with which it forms a rather stable complex. Removal of a substantial amount of this deoxyribonucleotide by treatment with DNAase I has no effect on the enzyme activity. The beta-lactamase has a wide spectrum of activity. Penicillins and delta 3-cephalosporins can be either good or poor substrates. Oxacillin, which is a poor substrate of most beta-lactamases from Gram-positive bacteria, is a good substrate of the beta-lactamase of Actinomadura R39. Its best substrate, however, is nitrocefin (kcat/Km: 2300 000 M-1.s-1; catalytic centre activity: 210 s-1). The kcat/Km values observed with some penicillins and delta 3-cephalosporins are similar to the values of the bimolecular rate constants that govern the formation of the acyl-enzyme intermediates between these antibiotics and the serine D-alanyl-D-alanine peptidase that is also secreted by the same strain Actinomadura R39. Such a relationship, however, is not observed with all the beta-lactam compounds tested. [less ▲]

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See detailStructure of a Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase at 2.5 A resolution.
Dideberg, O.; Charlier, Paulette ULg; Dive, Georges ULg et al

in Nature (1982), 299(5882), 469-470

Bacteria possess proteases that are specific for the peptide bonds between D-alanine residues, one of which has a free alpha-carboxyl group. These D-alanyl-D-alanine peptidases catalyse carboxypeptidation ... [more ▼]

Bacteria possess proteases that are specific for the peptide bonds between D-alanine residues, one of which has a free alpha-carboxyl group. These D-alanyl-D-alanine peptidases catalyse carboxypeptidation and transpeptidation reactions involved in bacterial cell wall metabolism1,2, and are inactivated by beta-lactam antibiotics. We have now elucidated the structure, at 2.5 Å resolution, of the penicillin-resistant Zn2+-containing D-alanyl-D-alanine peptidase of Streptomyces albus (Zn2+ G peptidase)3,4. The enzyme is shown to consist of two globular domains, connected by a single link. The N-terminal domain has three alpha-helices, and the C-terminal domain has three alpha-helices and five beta-strands. The Zn2+ ion is ligated by three histidine residues, and located in a cleft in the C-terminal domain. The mechanism of action of the enzyme may be related to that of other carboxypeptidases, which also contain functional Zn2+ ions. [less ▲]

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See detailConformational analysis of peptide substrates and inhibitors of the Zn2+ G and serine R61 D-alanyl-D-alanine peptidases
De Coen, Jean-Louis; Lamotte, Josette ULg; Ghuysen, Jean-Marie ULg et al

in European Journal of Biochemistry (1981), 121(1), 221-232

The tripeptide Nα,Nɛ-diacetyl-l-lysyl-d-alanyl-d-alanine (Ac2- l-LLys1-dAIa2-dAIa3), which is the standard substrate of the Zn2+ G and serine R61 d-alanyl-d-alanine peptidases, and several ldd tripeptide ... [more ▼]

The tripeptide Nα,Nɛ-diacetyl-l-lysyl-d-alanyl-d-alanine (Ac2- l-LLys1-dAIa2-dAIa3), which is the standard substrate of the Zn2+ G and serine R61 d-alanyl-d-alanine peptidases, and several ldd tripeptide analogues where the size and/or the electrical charge of the side chains at position 1, 2 or 3 have been modified (alterations affecting more than one position at the same time were not investigated) have been submitted to conformational analyses based on both short-range and long-range interactions. Among the many backbone conformers of minimal energy of the øii space that have been characterized, four types of conformers are the most probable ones. Depending on the peptides, these conformers may have varying relative probability P values so that the leader conformer is not always the same, but, in all cases, the sum of their P values is 90% or more. With the Gly1, Gly2 or Gly3 analogues (which encompass a larger conformational space), the above ∑P values are still as high as 35–50%. All the above tripeptides bind to the serine d-alanyl-d-alanine peptidase and with the exception of the Gly3 and Gly2 analogues, to the Zn2+d-alanyl-d-alanine peptidase with virtually the same efficacy, at least within a range of variation of the Km values for the substrates or the Ki values for the inhibitors, which is less than one order of magnitude. Structural variations at position 1, 2 or 3 in the peptides that are compatible with efficient binding are not necessarily compatible with substrate activity, thus converting the modified peptides into competitive inhibitors. In particular, substrate activity requires a long side chain at position 1 in the peptides. Conformational analyses of Ac2-lLys-dAla-dAla show that the main backbone has a tendency to adopt a ring-like shape from which the lLYS side chain protrudes as an extended structure. This latter structure forms with the C-terminal d-alanyl-d-alanine an angle varying between 120° and 180° (depending on the conformers) so that its N-terminal acetyl group is about 1–1.5 nm apart from the scissile amide bond. High turnover numbers (at enzyme saturation) also require a dAla at position 2 with both d-alanyl-d-alanine peptidases and at position 3 in the case of the serine d-alanyl-d-alanine peptidase. Finally, all the conformers of the lAla2 and lAla3 analogues of Ac2-lLys-dAla-dAla fall outside the backbone conformational space that comprises the φiφi angles exhibited by the four types of conformers of the ldd tripeptides. The lAla2 and lAla3 tripeptide analogues do not bind to the serine d-alanyl-d-alanine peptidase (at least at a 10 mM concentration) but they behave as noncompetitive inhibitors of the Zn2+d-alanyl-d-alanine peptidase. [less ▲]

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See detailOn the DD-carboxypeptidase enzyme system of Streptomyces strain K15
Leyh-Bouille, Mélina; Nguyen-Distèche, Martine ULg; Ghuysen, Jean-Marie ULg

in European Journal of Biochemistry (1981), 115(3), 579-584

Streptomyces K15 possesses a set of exocellular and cell-bound D-alanyl-D-alanine carboxypeptidases. Four of them have been isolated to the stage where each enzyme preparation contains on single ... [more ▼]

Streptomyces K15 possesses a set of exocellular and cell-bound D-alanyl-D-alanine carboxypeptidases. Four of them have been isolated to the stage where each enzyme preparation contains on single penicillin-binding protein. The exocellular 54000-Mr enzyme is extremely sensitive to benzylpenicillin and performs low transpeptidase activity on the carbonyl-donor/amino-acceptor tetrapeptide ACLLys(Gly)-DAla-DAla. The exocellular 40 000-Mr enzyme and the two lysozyme-releasable 40 000-Mr and 38 000-Mr enzymes are moderately sensitive to benzylpenicillin and have a high propensity to catalyse dimer formation from the aforementioned tetrapeptide monomer. [less ▲]

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See detailPrimary structure of the wall peptidoglycan of leprosy-derived corynebacteria
Janczura, E.; Leyh-Bouille, Mélina; Cocito, C. et al

in Journal of Bacteriology (1981), 145(2), 775-779

The cell walls isolated from axenically grown leprosy-derived corynebacteria were submitted to various chemical and enzymatic degradations. The glycan strands of the wall peptidoglycan are essentially ... [more ▼]

The cell walls isolated from axenically grown leprosy-derived corynebacteria were submitted to various chemical and enzymatic degradations. The glycan strands of the wall peptidoglycan are essentially composed of N-acetylglycosaminyl-N-acetylmuramic acid disaccharide units. Small amounts of N-acetylglycosaminyl-N-glycolylmuramic acid (less than 10%) were also detected. The muramic acid residues of adjacent glycan strands are substituted by amidated tetrapeptide units which, in turn, are cross-linked through direct linkages extending between the C-terminal D-alanine residue of one tetrapeptide and the mesodiaminopimelic acid residue of another tetrapeptide. Such a structure is very similar to that of the wall peptidoglycan found in the taxonomically related microorganisms of the Corynebacterium, Mycobacterium, and Nocardia groups. [less ▲]

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See detailPenicillins and Δ3-cephalosporins as inhibitors and mechanism-based inactivators of D-alanyl-D-Ala peptidases
Ghuysen, Jean-Marie ULg; Frère, Jean-Marie ULg; Leyh-Bouille, Mélina et al

in Burgen, A. S. V.; Roberts, G. C. K. (Eds.) Topics in molecular pharmacology (1981)

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See detailInteractions between non-classical β-lactam compounds and the β-lactamases of Actinomadura R39 and Streptomyces albus G
Kelly, Judith; Frère, Jean-Marie ULg; Duez, Colette ULg et al

in Biochemical Journal (1981), 199

Streptomyces albus G secretes a Zn2+-containing D-alanyl-D-alanine peptidase. Streptomyces R61 and Actinomadura R39 secrete D-alanyl-D-alanine-cleaving serine peptidases. The effect of non-classical beta ... [more ▼]

Streptomyces albus G secretes a Zn2+-containing D-alanyl-D-alanine peptidase. Streptomyces R61 and Actinomadura R39 secrete D-alanyl-D-alanine-cleaving serine peptidases. The effect of non-classical beta-lactam antibiotics on these three model enzymes has been studied. Mecillinam, cefoxitin, quinacillin, quinacillin sulphone, clavulanate and N-formimidoylthienamycin have no effect on the Zn2+-containing enzyme. 6-Amino-penicillanic acid slowly inactivates this enzyme and 7-aminocephalosporanic acid behaves as a reversible inhibitor. Cefoxitin and N-formimidoylthienamycin are potent anti-bacterial agents; they effectively inactivate the serine R39 enzyme and, to a lesser extent, the serine R61 enzyme. All the other beta-lactam compounds tested, including mecillinam, are slow inactivators of these serine enzymes. The intermediates formed between 6-aminopenicillanic acid and the R61 and R39 enzymes are long- and short-lived respectively, whereas those formed between 7-aminocephalosporanic acid and the same R61 and R39 enzymes are short- and long-lived respectively. Breakdown of the short-lived intermediates thus obtained gives rise to several ninhydrin-positive degradation products. The intermediates formed between clavulanate and the serine enzymes are long-lived. With the R39 enzyme, the inactivated complex formed in a first step undergoes subsequent monomolecular rearrangement to give rise to a second species exhibiting a high absorbance at 273 nm. [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 detailPenicillin binding proteins and model transpeptidase activity of the plasma membranes of Streptomyces strains R61 and rimosus
Dusart, Jean; Reynolds, Peter E.; Ghuysen, Jean-Marie ULg

in FEMS Microbiology Letters (1981), 12(3), 299-303

Streptomyces R61 and S. rimosus have an atypical penicillin-binding protein (PBP) pattern characterized by a large amt. of a 25,000-mol.-wt. protein and a small amt. of a 50,000-mol.-wt. protein. The 25 ... [more ▼]

Streptomyces R61 and S. rimosus have an atypical penicillin-binding protein (PBP) pattern characterized by a large amt. of a 25,000-mol.-wt. protein and a small amt. of a 50,000-mol.-wt. protein. The 25,000-mol.-wt. PBP exhibits high thermostability and apparently is the membrane-bound enzyme which catalyzes the model transpeptidase reaction in which Ac2-L-Lys-D-Ala-D-Ala serves as the carbonyl donor and Gly-Gly as the amino acceptor. Whether the thermolabile 50,000-mol.-wt. PBP is also a transpeptidase and is degraded into small fragments or converted into the 25,000-mol.-wt. PBP is unclear. [on SciFinder(R)] [less ▲]

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See detailStudies on the primary structures of the exocellular D-Alanyl-D-Alanine peptidases of Streptomyces strain R61 and Actinomadura strain R39
Duez, Colette ULg; Frère, Jean-Marie ULg; Ghuysen, Jean-Marie ULg et al

in Biochimica et Biophysica Acta - General Subjects (1981), 671

The Mr 37 000 D-alanyl-D-alanine peptidase excreted by Streptomyces R61 and the Mr 53 000 D-alanyl-D-alanine peptidase excreted by Actinomadura R39 are both characterized by a very uneven distribution of ... [more ▼]

The Mr 37 000 D-alanyl-D-alanine peptidase excreted by Streptomyces R61 and the Mr 53 000 D-alanyl-D-alanine peptidase excreted by Actinomadura R39 are both characterized by a very uneven distribution of the basic (Arg + Lys) amino acid residues. Trypsin degradation of the heat-denatured enzymes generates (1) thirteen soluble peptides which contain from 2 to 28 residues in the case of the R61 enzyme and nineteen soluble peptides which contain 2 to 39 residues in the case of the R39 enzyme; and (2) three large segments or core peptides which, irrespective of the enzymes from which they originate, consist of 50-60, 70-80 and 110-120 residues. About 90% of the basic (Arg + Lys) amino acid residues are recovered in the soluble tryptic peptides. The core peptides represent 62% (Mr approximately 23 000) and 45% (Mr approximately 24 000) of the untreated R61 and R39 enzymes, respectively. One 28-residue soluble peptide isolated from the R61 enzyme represents the N-terminal portion of the protein whose sequence has been established. The penicillin attachment site of the R61 enzyme has been located in one of the core peptides. For the R39 enzyme, indirect evidence shows that the penicillin binding site is probably within one of the soluble peptides. [less ▲]

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See detailThe penicillin-binding site in the exocellular DD-carboxypeptidase-transpeptidase of Actinomadura R39
Duez, Colette ULg; Joris, Bernard ULg; Frère, Jean-Marie ULg et al

in Biochemical Journal (1981), 193

Heat denaturation and Pronase degradation of the complex previously formed between benzylpenicillin and the exocellular DD-carboxypeptidase-transpeptidase of Actinomadura R39 yields a heptapeptide H-Leu ... [more ▼]

Heat denaturation and Pronase degradation of the complex previously formed between benzylpenicillin and the exocellular DD-carboxypeptidase-transpeptidase of Actinomadura R39 yields a heptapeptide H-Leu-Pro-Ala-Ser-Asn-Gly-Val-OH, where the benzylpenicilloyl group is ester-linked to the serine residue. This linkage is very labile and its hydrolysis causes the release of benzylpenicilloate. In contrast, the native benzylpenicilloyl-enzyme complex is very stable (half-life 70h at 370C) and its breakdown proceeds via fragmentation of the bound benzylpenicilloyl group [Fuad, Frere, Ghuysen, Duez & Iwatsubo (1976) Biochem. J. 155, 623-6291. [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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