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M Nguyen-Distèche

Publications and source records attributed to M Nguyen-Distèche.

36 records · Page 2Linked to original sources
PubMed1988

Overexpression, solubilization and refolding of a genetically engineered derivative of the penicillin-binding protein 3 of Escherichia coli K12.

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.

Acyltransferases↗
PubMed1987

The pH dependence of the active-site serine DD-peptidase of Streptomyces R61.

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.

Acylation↗
PubMed1987

Effects of thiol reagents on Streptomyces K15 DD-peptidase-catalysed reactions.

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.

Chloromercuribenzoates↗
PubMed1986

Active-site-serine D-alanyl-D-alanine-cleaving-peptidase-catalysed acyl-transfer reactions. Procedures for studying the penicillin-binding proteins of bacterial plasma membranes.

Under certain conditions, the values of the parameters that govern the interactions between the active-site-serine D-alanyl-D-alanine-cleaving peptidases and both carbonyl-donor substrates and beta-lactam suicide substrates can be determined on the basis of the amounts of (serine ester-linked) acyl-protein formed during the reactions. Expressing the 'affinity' of a beta-lactam compound for a DD-peptidase in terms of second-order rate constant of enzyme acylation and first-order rate constant of acyl-enzyme breakdown rests upon specific features of the interaction (at a given temperature) and permits study of structure-activity relationships, analysis of the mechanism of intrinsic resistance and use of a 'specificity index' to define the capacity of a beta-lactam compound of discriminating between various sensitive enzymes. From knowledge of the first-order rate constant of acyl-enzyme breakdown and the given time of incubation, the beta-lactam compound concentrations that are necessary to achieve given extents of DD-peptidase inactivation can be converted into the second-order rate constant of enzyme acylation. The principles thus developed can be applied to the study of the multiple penicillin-binding proteins that occur in the plasma membranes of bacteria.

Acylation↗
PubMed1986

Streptomyces K15 DD-peptidase-catalysed reactions with ester and amide carbonyl donors.

In water, the purified 26 000-Mr membrane-bound DD-peptidase of Streptomyces K15 hydrolyses the ester carbonyl donor Ac2-L-Lys-D-Ala-D-lactate (release of D-lactate) and the amide carbonyl donor Ac2-L-Lys-D-Ala-D-Ala (release of D-alanine) with accumulation of acyl- (Ac2-L-Lys-D-alanyl-)enzyme. Whereas hydrolysis of the ester substrate proceeds to completion, hydrolysis of the amide substrate is negligible because of the capacity of the K15 DD-peptidase for utilizing the released D-alanine in a transfer reaction (Ac2-L-Lys-D-Ala-D-Ala + D-Ala----Ac2-L-Lys-D-Ala-D-Ala + D-Ala) that maintains the concentration of the amide substrate at a constant level. In the presence of an amino acceptor X-NH2 (Gly-Gly or Gly-L-Ala) related to the Streptomyces peptidoglycan, both amide and ester carbonyl donors are processed without detectable accumulation of acyl-enzyme. Under proper conditions, the acceptor activity of water and, in the case of the amide substrate, the acceptor activity of the released D-alanine can be totally overcome so that the two substrates are quantitatively converted into transpeptidated product Ac2-L-Lys-D-Ala-NH-X (and hydrolysis is prevented). Experimental evidence suggests that the amino acceptor modifies both the binding of the carbonyl donor to the enzyme and the ensuing rate of enzyme acylation.

Acylation↗
PubMed1986

Streptomyces K15 DD-peptidase-catalysed reactions with suicide beta-lactam carbonyl donors.

The values of the kinetic parameters that govern the interactions between the Streptomyces K15 DD-peptidase and beta-lactam compounds were determined by measuring the inactivating effect that these compounds exert on the transpeptidase activity of the enzyme and, in the case of [14C]benzylpenicillin and [14C]cefoxitin, by measuring the amounts of acyl-enzyme formed during the reaction. K15 DD-peptidase binds benzylpenicillin or cefoxitin at a molar ratio of 1:1. Benzylpenicilloate is the major product released during breakdown of the acyl-enzyme formed with benzylpenicillin. Benzylpenicillin is not a better acylating agent than the amide Ac2-L-Lys-D-Ala-D-Ala and ester Ac2-L-Lys-D-Ala-D-lactatecarbonyl-donor substrates. beta-Lactam compounds possessing a methoxy group on the alpha-face of the molecule show high inactivating potency.

Acylation↗
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↗
PubMed1982

Isolation 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.

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.

Bacterial Proteins↗
PubMed1982

Penicillin-binding proteins and carboxypeptidase/transpeptidase activities in Proteus vulgaris P18 and its penicillin-induced stable L-forms.

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).

Bacterial Proteins↗
PubMed1981

On the DD-carboxypeptidase enzyme system of Streptomyces strain K15.

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.

Carboxypeptidases↗
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↗