References of "Ghuysen, Jean-Marie"
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See detailNumerical computation of the electrostatic interaction energy between methanol and the dyad water-imidazole
Dehareng, Dominique ULg; Dive, Georges ULg; Lamotte-Brasseur, Josette ULg et al

in Theoretica Chimica Acta (1989), 76(2), 85-94

The electrostatic interaction energy between methanol and the dyad water-imidazole has been computed numerically at three levels of approximation from 3D grids of the charge density of one partner and the ... [more ▼]

The electrostatic interaction energy between methanol and the dyad water-imidazole has been computed numerically at three levels of approximation from 3D grids of the charge density of one partner and the electrostatic potential of the other. The minimum positions and energy values thus obtained compare well with those calculated analytically. The numerical procedure is especially interesting for the prediction of the stable conformers. [less ▲]

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See detailCrystallographic mapping of β-lactams bound to a D-alanyl-D-alanine peptidase target enzyme
Kelly, Judith A.; Knox, James R.; Zhao, Haiching C. et al

in Journal of Molecular Biology (1989), 209(2), 281-295

X-ray crystallography has been used to examine the binding of three members of the beta-lactam family of antibiotics to the D-alanyl-D-alanine peptidase from Streptomyces R61, a target of penicillins ... [more ▼]

X-ray crystallography has been used to examine the binding of three members of the beta-lactam family of antibiotics to the D-alanyl-D-alanine peptidase from Streptomyces R61, a target of penicillins. Cephalosporin C, the monobactam analog of penicillin G and (2,3)-alpha-methylene benzylpenicillin have been mapped at 2.3 A resolution in the form of acyl-enzyme complexes bound to serine 62. On the basis of the positions of these inhibitors, the binding of a tripeptide substrate for the enzyme, L-lysyl-D-alanyl-D-alanine, has been modeled in the active site. The binding of both inhibitors and substrate is facilitated by hydrogen-bonding interactions with a conserved beta-strand (297-303), which is antiparallel to the beta-lactam's acylamide linkage or the substrate's peptide bond. The active site is similar to that in beta-lactamases. [less ▲]

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See detailCloning and amplified expression in Streptomyces lividans of the gene encoding the extracellular β-lactamase of Actinomadura R39
Fraipont, Claudine ULg; Duez, Colette ULg; Matagne, André ULg et al

in Biochemical Journal (1989), 262(3), 849-854

By using the promoter-probe plasmid pIJ424, genomic DNA fragments of Actinomadura R39 were shown to have promoter activity in Streptomyces lividans. The same 100-200-copy-number plasmid was used to clone ... [more ▼]

By using the promoter-probe plasmid pIJ424, genomic DNA fragments of Actinomadura R39 were shown to have promoter activity in Streptomyces lividans. The same 100-200-copy-number plasmid was used to clone in S. lividans TK24, the gene that encodes the Actinomadura R39, β-lactamase. Gene cloning resulted in an amplified expression of the , β lactamase when compared with the amounts of enzyme produced by the original strain (1 mg versus 0.008 mg . litre of culture-1). [less ▲]

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See detailThe Streptomyces K15 DD-peptidase/penicillin-binding protein. Active site and sequence of the N-terminal region.
Leyh-Bouille, Mélina; Vanbeeumen, Jozef; Renier-Pirlot, Suzanne et al

in Biochemical Journal (1989), 260(2), 601-604

The N-terminal region of the Streptomyces K15 DD-peptidase/penicillin-binding protein shows high homology with that of other penicillin-interactive proteins or domains. The active-site serine residue of ... [more ▼]

The N-terminal region of the Streptomyces K15 DD-peptidase/penicillin-binding protein shows high homology with that of other penicillin-interactive proteins or domains. The active-site serine residue of the conserved tetrad Ser-Xaa-Xaa-Lys occurs at position 35. There is no indication for the presence of a signal peptide or an N-terminal hydrophobic sequence, suggesting that the Streptomyces K15 enzyme is probably anchored to the membrane by a C-terminal peptide segment. [less ▲]

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See detailUsing X-ray diffraction results and computer graphics to design beta-lactams
Kelly, Judith A.; Knox, J. R.; Moews, P. C. et al

in Kansenshogaku Zasshi = Journal of the Japanese Association for Infectious Diseases (1988), 62 Suppl

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See detailThe Active-Site-Serine Penicillin-Recognizing Enzymes as Members of the Streptomyces R61 Dd-Peptidase Family
Joris, Bernard ULg; Ghuysen, Jean-Marie ULg; Dive, Georges ULg et al

in Biochemical Journal (1988), 250(2), 313-324

Homology searches and amino acid alignments, using the Streptomyces R61 DD-peptidase/penicillin-binding protein as reference, have been applied to the beta-lactamases of classes A and C, the Oxa-2 beta ... [more ▼]

Homology searches and amino acid alignments, using the Streptomyces R61 DD-peptidase/penicillin-binding protein as reference, have been applied to the beta-lactamases of classes A and C, the Oxa-2 beta-lactamase (considered as the first known member of an additional class D), the low-Mr DD-peptidases/penicillin-binding proteins (protein no. 5 of Escherichia coli and Bacillus subtilis) and penicillin-binding domains of the high-Mr penicillin-binding proteins (PBP1A, PBP1B, PBP2 and PBP3 of E. coli). Though the evolutionary distance may vary considerably, all these penicillin-interactive proteins and domains appear to be members of a single superfamily of active-site-serine enzymes distinct from the classical trypsin or subtilisin families. The amino acid alignments reveal several conserved boxes that consist of strict identities or homologous amino acids. The significance of these boxes is highlighted by the known results of X-ray crystallography, chemical derivatization and site-directed-mutagenesis experiments. [less ▲]

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See detailCharacterization of the Trypsin-Solubilized Penicillin-Binding Proteins of Enterococcus hirae (Streptococcus faecium)
El Kharroubi, Aboubaker; Jacques, Philippe; Piras, Graziella et al

in Actor, Paul; Daneo-Moore, Lolita; Higgins, Michael L. (Eds.) et al Antibiotic Inhibition of Bacterial Cell Surface Assembly and Function (1988)

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See detailProperties and crystallization of a genetically engineered, water-soluble derivative of penicillin-binding protein 5 of Escherichia coli K12
Ferreira, Luis C; Schwarz, Uli; Keck, Wolfgang et al

in European Journal of Biochemistry (1988), 171(1-2), 11-16

Derivatives of the Escherichia coli penicillin-binding protein 5 (PBP5) with truncated carboxyl terminals were obtained by altering the carboxyl-coding end of the dacA gene. After cloning the modified ... [more ▼]

Derivatives of the Escherichia coli penicillin-binding protein 5 (PBP5) with truncated carboxyl terminals were obtained by altering the carboxyl-coding end of the dacA gene. After cloning the modified dacA gene into a runaway-replication-control plasmid, one clone that overproduced and excreted the desired protein into the periplasm was used as a source for the isolation of a water-soluble PBP5 (i.e. PBP5S). In PBP5S the carboxyl-terminal 21-amino-acid region of the wild-type protein was replaced by a short 9-amino-acid segment. Milligram amounts of PBP5S were purified by penicillin affinity chromatography in the absence of detergents or of chaotropic agents. PBP5S was stable and possessed DD-carboxypeptidase activity without added Triton X-100. Upon reaction with [14C]benzylpenicillin it was converted into a rather short-lived acyl-enzyme complex, as observed with PBP5. Both PBP5 and PBP5S were crystallized. In contrast to PBP5, PBP5S yielded enzymatically active, well-formed prismatic crystals suitable for X-ray analysis. [less ▲]

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See detailPurification and Characterization of a β-lactam-Resistant Penicillin-Binding Protein from Enterococcus hirae (Streptococcus faecium)
Jacques, Philippe; El Kharroubi, Aboubaker; Joris, Bernard ULg et al

in Actor, Paul; Daneo-Moore, Lolita; Higgins, Michael L. (Eds.) et al Antibiotic Inhibition of Bacterial Cell Surface Assembly and Function (1988)

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See detailPenicillin-recognizing enzymes
Frère, Jean-Marie ULg; Joris, Bernard ULg; Dideberg, Otto et al

in Biochemical Society Transactions (1988), 16(6), 934-938

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See detailBacterial Active-Site Serine Penicillin-Interactive Proteins and Domains: Mechanism, Structure, and Evolution
Ghuysen, Jean-Marie ULg

in Reviews of Infectious Diseases (1988), 10(4), 726-732

The bacterial active-site serine penicillin-hydrolyzing and penicillin-binding proteins or domains operate by a serine-ester-linked acyl enzyme mechanism similar to that of the peptidases of the trypsin ... [more ▼]

The bacterial active-site serine penicillin-hydrolyzing and penicillin-binding proteins or domains operate by a serine-ester-linked acyl enzyme mechanism similar to that of the peptidases of the trypsin and subtilisin families. On the basis of known primary and tertiary structures, predictive studies support the view that these proteins or domains form a superfamily of evolutionarily related enzymes. Although--depending on the evolutionary distance--they may have very different amino acid sequences and distinct functional characteristics and specificities, they all would have conserved the same unprecedented type of polypeptide scaffolding. When obtained, complete structural information should provide the necessary tools for the rational design of novel types of inactivators of these important enzyme targets. [less ▲]

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See detailBiochemistry of beta-lactamases and penicillin-binding proteins - Discussion
Curtis, N. A. C.; Bennett, P. M.; Nicholas, R. et al

in Reviews of Infectious Diseases (1988), 10(4), 739-742

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See detailOverexpression, solubilization and refolding of a genetically engineered derivative of the penicillin-binding protein 3 of Escherichia coli K12.
Bartholomé-De Belder, J.; Nguyen-Distèche, Martine ULg; Houba-Herin, N. et al

in Molecular Microbiology (1988), 2(4), 519-525

Replacement of the amino-terminal 40-amino-acid region of the 588-amino-acid precursor of the membrane-bound penicillin-binding protein 3 (PBP3) by the decapeptide MKGKEFQAWI was carried out by altering ... [more ▼]

Replacement of the amino-terminal 40-amino-acid region of the 588-amino-acid precursor of the membrane-bound penicillin-binding protein 3 (PBP3) by the decapeptide MKGKEFQAWI was carried out by altering the amino-coding end of the ftsI gene. Insertion of the modified gene into a runaway-replication plasmid under the control of a fused lpp promoter and lac promoter/operator, resulted in the overexpression by Escherichia coli of the modified PBP3 (designated PBP3**) in the cytoplasm. About 80% of the accumulated PBP3** underwent sequestration in the form of insoluble protein granules that were isolated by cell breakage or cell lysis. After selective removal of contaminants by an EDTA-lysozyme/DNase (deoxyribonuclease)/Nonidet extraction, treatment of the granules with guanidinium chloride followed by dialysis against buffer containing 0.5 M NaCl yielded a refolded, water-soluble PBP3**, which, upon chromatography on Superose 12, exhibited the expected 60,000 molecular mass. The refolded PBP3** bound benzylpenicillin in a 1 to 1 molar ratio, was highly sensitive to aztreonam and showed the same degree of thermostability, in terms of penicillin-binding capacity, as the parent, membrane-bound PBP3, suggesting that protein refolding occurred with formation of the correct intramolecular interactions. Two to three mg of refolded PBP3** can be obtained from 1 litre of culture of the overproducing strain. [less ▲]

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See detailThe importance of the negative charge of β-lactam compounds for the inactivation of the active-site serine DD-peptidase of Streptomyces R61
Varetto, Louis ULg; Frère, Jean-Marie ULg; Ghuysen, Jean-Marie ULg

in FEBS Letters (1987), 225(1-2), 218-222

The interaction between the Streptomyces R61 penicillin-sensitive DD-peptidase and deacetyl-cephalosporin C or its lactone derivative has been studied at different pH values. The results show the ... [more ▼]

The interaction between the Streptomyces R61 penicillin-sensitive DD-peptidase and deacetyl-cephalosporin C or its lactone derivative has been studied at different pH values. The results show the importance of an enzyme group of pK approximately equal to 9 which might form an ion pair with the free carboxylate of the former compound. This electrostatic interaction is shown to contribute to the formation of the first, non-covalent enzyme-inactivator complex by a factor of at least 50. [less ▲]

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See detailCloning and amplified expression in Streptomyces lividans of the gene encoding the extracellular β-lactamase from Streptomyces cacaoi
Lenzini, Mauro V; Nojima, Shiego; Dusart, Jean et al

in Journal of General Microbiology (1987), 133(10), 2915-2920

A 19 kb SphI DNA fragment containing the gene for the extracellular active-site serine beta-lactamase of Streptomyces cacaoi KCC-SO352 was cloned in Streptomyces lividans TK24 using the high-copy-number ... [more ▼]

A 19 kb SphI DNA fragment containing the gene for the extracellular active-site serine beta-lactamase of Streptomyces cacaoi KCC-SO352 was cloned in Streptomyces lividans TK24 using the high-copy-number plasmid pIJ702 as vector. A 30-fold higher yield of beta-lactamase was obtained from S. lividans strain ML1, carrying the recombinant plasmid pDML51, than from S. cacaoi grown under optimal production conditions. In all respects (molecular mass, isoelectric point, kinetics of inhibition by beta-iodopenicillanate) the overproduced S. lividans ML1 beta-lactamase was identical to the original S. cacaoi enzyme. A considerable reduction of beta-lactamase production was caused by elimination of a 12.8 kb portion of the 19 kb DNA fragment by cleavage at an internal SphI site located more than 3 kb upstream of the beta-lactamase structural gene. The beta-lactamase gene was located within a 1.8 NcoI-BclI fragment but when this fragment was cloned in S. lividans pIJ702, the resulting strain produced hardly any more beta-lactamase than the original S. cacaoi. [less ▲]

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See detailNucleotide sequence of the gene encoding the Streptomyces albus G β-lactamase precursor
Dehottay, Philippe; Dusart, Jean; De Meester, Fabien et al

in European Journal of Biochemistry (1987), 166(2), 345-350

A 1400-base DNA fragment, which contains the gene encoding the extracellular active-site serine beta-lactamase of Streptomyces albus G previously cloned into Streptomyces lividans [Dehottay et al. (1986 ... [more ▼]

A 1400-base DNA fragment, which contains the gene encoding the extracellular active-site serine beta-lactamase of Streptomyces albus G previously cloned into Streptomyces lividans [Dehottay et al. (1986) Gene 42, 31-36], was sequenced. The gene codes for a 314-amino-acid precursor, the N-terminal region of which has the characteristics of a signal peptide. The beta-lactamase as excreted by the host strain S. lividans PD6 has a ragged N-terminus, indicating either the presence of a leader peptidase of poor specificity or the action of an aminopeptidase. The primary structure (as deduced from the nucleotide sequence) was confirmed by amino acid sequencing of a 16-residue stretch at the amino terminus of the protein, a 12-residue stretch containing the active-site serine [De Meester et al. (1987) Biochem. J. 244, 427-432] and a 23-residue stretch obtained by trypsin digestion of the protein. The beta-lactamase belongs to class A, has three half-cystine residues (one of which occurs on the amino side of the active-site serine) and is inactivated by thiol reagents. Putative ribosome binding site and terminator region were identified. [less ▲]

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See detailThe pH dependence of the active-site serine DD-peptidase of Streptomyces R61
Varetto, Louis; Frère, Jean-Marie ULg; Nguyen-Distèche, Martine ULg et al

in European Journal of Biochemistry (1987), 162(3), 525-531

Titration of the active-site serine DD-peptidase of Streptomyces R61 shows that formation of acyl enzyme during hydrolysis of the substrate Ac2-L-Lys-D-Ala-D-Ala and enzyme inactivation by the beta-lactam ... [more ▼]

Titration of the active-site serine DD-peptidase of Streptomyces R61 shows that formation of acyl enzyme during hydrolysis of the substrate Ac2-L-Lys-D-Ala-D-Ala and enzyme inactivation by the beta-lactam compounds benzylpenicillin, N-acetylampicillin and ampicillin relies on the acidic form of an enzyme's group of pK approximately equal to 9.5. It is proposed that protonation of a lysine epsilon-amino group facilitates initial binding by charge pairing with the free carboxylate of the substrate and the beta-lactam molecules. Lowering the pH from 7 to 5 has no effect on the second-order rate constant of enzyme acylation by benzylpenicillin and N-acetylampicillin but results in a decreased rate constant of acylation by ampicillin and Ac2-L-Lys-D-Ala-D-Ala. Protonation of the side-chain amino group of ampicillin and a decreased efficacy of the initial binding of the peptide to the enzyme seem to be responsible for the observed effects. Whatever the molecule bound to the enzyme, there is no sign for the active involvement of an enzyme's histidine residue of pK 6.5-7.0 in the hydrolysis pathway. [less ▲]

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See detailPrimary structure of the Streptomyces R61 extracellular DD-peptidase. 2. Amino acid sequence data
Joris, Bernard ULg; Jacques, Philippe; Frère, Jean-Marie ULg et al

in European Journal of Biochemistry (1987), 162(3), 519-524

In order to confirm the Streptomyces codon usage, the Streptomyces R61 DD-peptidase was fragmented by cyanogen bromide cleavage of the carboxymethylated protein, trypsin digestion of the carboxymethylated ... [more ▼]

In order to confirm the Streptomyces codon usage, the Streptomyces R61 DD-peptidase was fragmented by cyanogen bromide cleavage of the carboxymethylated protein, trypsin digestion of the carboxymethylated protein and trypsin digestion of the protein treated with beta-iodopenicillinate and endoxo-delta 4-tetrahydrophthalic acid. The isolated peptides, which altogether represented more than 50% of the polypeptide chain, were sequenced. The data thus obtained were in excellent agreement with the primary structure of the protein as deduced from the nucleotide sequence of the cloned gene. Though a weak acylating agent, beta-iodopenicillanate reacted selectively with the active site of the DD-peptidase and formed an adduct which mas much more stable than that formed with benzylpenicillin, thus facilitating the isolation and characterization of the active-site peptide. [less ▲]

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See detailEffects of thiol reagents on Streptomyces K15 DD-peptidase-catalysed reactions
Leyh-Bouille, Mélina; Nguyen-Distèche, Martine ULg; Bellefroid-Bourguignon, Catherine et al

in Biochemical Journal (1987), 241(3), 893-897

The 26,000-Mr DD-peptidase of Streptomyces K15 binds one equivalent of thiol reagents as 5,5'-dithiobis-(2-nitrobenzoate) or p-chloromercuribenzoate (pCMB). Derivatization of the DD-peptidase by pCMB ... [more ▼]

The 26,000-Mr DD-peptidase of Streptomyces K15 binds one equivalent of thiol reagents as 5,5'-dithiobis-(2-nitrobenzoate) or p-chloromercuribenzoate (pCMB). Derivatization of the DD-peptidase by pCMB decreases the efficacy of the initial binding of the ester carbonyl donor Ac2-L-Lys-D-Ala-D-lactate to the enzyme (K), the rate of enzyme acylation by the donor (K+2) and the rate of enzyme deacylation (k+3). However, the value of the k+2/k+3 ratio, and therefore the percentage of total enzyme which, at saturating concentrations of the donor, is present as acyl-enzyme at the steady state of the reaction, are not modified. The enzyme's binding sites for pCMB and benzylpenicillin are not mutually exclusive. But, when compared with the native enzyme, the pCMB-derivatized enzyme undergoes acylation by benzylpenicillin with a decreased second-order-rate constant (k+2/K) value and gives rise to a penicilloyl adduct of increased stability. Since the acyl-enzyme mechanism is not annihilated by pCMB derivatization, it is proposed that basically, and like all the other DD-peptidases/penicillin-binding proteins so far characterized, the Streptomyces K15 DD-peptidase is an active-site-serine enzyme. [less ▲]

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