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J Dusart

Publications and source records attributed to J Dusart.

49 records · Page 3Linked to original sources
PubMed1984

The active site of the P99 beta-lactamase from Enterobacter cloacae.

Labelling the beta-lactamase of Enterobacter cloacae P99 with a poor substrate or a mechanism-based inactivator points to an active-site serine residue in a sequence closely resembling that of the ampC beta-lactamase. These results establish the P99 enzyme as a class-C beta-lactamase, and the concurrence of the two approaches helps to confirm the reliability of determining active-site sequences with the aid of mechanism-based inactivators.

Amino Acids↗
PubMed1984

Bacterial wall peptidoglycan, DD-peptidases and beta-lactam antibiotics.

Wall peptidoglycan expansion in bacteria rests upon a cytoplasmic D-Ala: D-Ala ligase (ADP) which catalyses synthesis of a D-Ala-D-Ala dipeptide (with accompanying hydrolysis of one molecule of ATP) and a set of DD-peptidases which utilize this D-Ala-D-Ala dipeptide--once it has been translocated at the outer face of the plasma membrane as the C-terminal portion of a disaccharide peptide unit--as carbonyl donor for transpeptidation and carboxypeptidation reactions (without additional energy expenditure). Four DD-peptidases have been selected which differ from each other with respect to the effects that amino compounds exert on the fate and rate of consumption of a D-Ala-D-Ala terminated amide carbonyl donor analogue. They serve as models to understand the different mechanisms by which the DD-peptidases perform catalysis and show widely varying responses to the action of beta-lactams, from extreme sensitivity to very high resistance.

Amino Acyl-tRNA Synthetases↗
PubMed1977

The peptidoglycan crosslinking enzyme system in Streptomyces strains R61, K15 and rimosus.

The DD-carboxypeptidase-transpeptidase enzyme system in Streptomyces strain K15 consists of: (1) a membrane-bound transpeptidase capable of performing low DD-carboxypeptidase activity; and (2) a set of DD-carboxypeptidases: (a) membrane-bound, (b) lysozyme-releasable and (c) exocellular, having low transpeptidase activities in aqueous media and at low acceptor concentrations. The DD-carboxypeptidases are related to each other and may belong to the same pathway leading to enzyme excretion. A similar enzyme system occurs in Streptomyces strain R61 except that the membrane-bound DD-carboxypeptidase activity is low when compared with the membrane-bound transpeptidase activity. In Streptomyces rimosus the enzyme system consists almost exclusively of the membrane-bound transpeptidase and the levels of membrane-bound, lysozyme-releasable and exocellular DD-carboxypeptidases are very low.

Carboxypeptidases↗
PubMed1977

The peptidoglycan crosslinking enzyme system in Streptomyces strains R61, K15 and rimosus. Kinetic coefficients involved in the interactions of the membrane-bound transpeptidase with peptide substrates and beta-lactam antibiotics.

The transpeptidation reaction performed by the membranes of Streptomyces strain R61 fits the general rate equation for an enzyme-catalysed bimolecular reaction. The same membranes (E) interact with beta-lactams (I) to form inactive penicillin-enzyme-membrane complexes (EI) of rather high stability, which subsequently break down (E + I leads to EI leads to E + degradation products). The enzyme is regenerated and the antibiotic is released in the form of an inactive metabolite. With benzylpenicillin, the degradation product is benzylpenicilloic acid. The reaction is heat-labile. The first step of the reaction (E + I leads to EI) is characterized by a second-order rate constant (kformation in M-1 s-1) and the second step (EI leads to E + degradation products) by a first-order rate constant (kbreakdown in s-1). The effects in vitro of various beta-lactams on the membrane-bound transpeptidase, as expressed by the relevant kformation and kbreakdown values, parallel the effects in vivo of the same antibiotics as expressed by their ability to prevent the germination and growth of conidiospores. The kinetic parameters of the transpeptidase that was solubilized with N-cetyl-N,N,N-trimethylammonium bromide with respect to its interaction with both peptide substrates and beta-lactam antibiotics are quantitatively different from those of the membrane-bound enzyme. Moreover, the solubilized enzyme fragments benzylpenicillin with formation of phenylacetylglycine, a reaction which is similar to that catalysed by the exocellular R61 enzyme. The membranes of Streptomyces strains rimosus and K15 possess an active 'classic' penicillinase. They were not studied but the kinetic coefficients of the corresponding solubilized transpeptidases were determined and compared with those of the solubilized enzyme from strain R61.

Carboxypeptidases↗
PubMed1975

The catalytic activity and penicillin sensitivity in the liquid and frozen states of membrane-bound and detergent-solubilised transpeptidase of Streptomyces R61.

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.

Acyltransferases↗
PubMed1973

DD-carboxypeptidase-transpeptidase and killing site of beta-lactam antibiotics in Streptomyces strains R39, R61, and K11.

Additional evidence is given that in Streptomyces strains R39, R61, and K11 the same enzyme performs dd-carboxypeptidase and transpeptidase activities and that this enzyme is the killing site of beta-lactam antibiotics. With strain R61, it was found that the exocellular enzyme has a sensitivity towards some antibiotics different from that of the membrane-bound enzyme. Under the growth conditions used in the present investigations, beta-lactamase activity was not involved in susceptibility to beta-lactam antibiotics.

Acyltransferases↗
PubMed1973

Exocellular beta-lactamases of Streptomyces albus G and strains R39 and K11.

The beta-lactamases excreted by the highly benzylpenicillin-susceptible Streptomyces strain R39 and the highly benzylpenicillin-resistant Streptomyces albus G were isolated and purified. Neither beta-lactamase exhibited dd-carboxypeptidase activity. Both were anionic at pH 8.3, did not require metal ions, and were not sensitive to iodine, but were inhibited by Cu(2+) and readily inactivated by heat. p-Chloromercuribenzoate, iodoacetate, p-aminobenzoate, and substrates and inhibitors of dd-carboxypeptidase had no effect on beta-lactamase activity. The K(m) and V(max) values for beta-lactamase activity were studied with 6-aminopenicillanic acid and with various penicillins and cephalosporins. The beta-lactamase from the related strain K11 of Streptomyces, which is intermediate in its susceptibility to benzylpenicillin, was partially purified, and its activity was compared on the various substrates.

Carboxypeptidases↗