Role of Residue Lys315 in the Mechanism of Action of the Enterobacter-cloacae 908r Beta-lactamase

The role of the highly conserved Lys315 residue in the catalytic mechanism of a class C beta-lactamase has been probed by site-directed mutagenesis. Lys315 has been replaced by a histidine in the Enterobacter cloacae 908R beta-lactamase, thus introducing a tritratabre group to probe the role of the positive charge, and by a glutamine. The effects of these mutations have been studied on the kinetics of penicillin G and cephalothin turnover and on the pre-steady-state kinetics with carbenicillin at different pH. Results showed that substrate binding was not impaired by the mutations, so that an interaction with the substrate-free carboxylate in the Henri-Michaelis complex could be ruled out. Lys315 must have a catalytic role as shown by the decreased acylation and deacylation rates observed with the mutant enzymes. The mutants exhibited a lower activity at acidic pH, and this observation could be correlated with a decreased affinity for (3-aminophenyl)boronate, a compound devoid of free carboxylate which binds to the active site and forms an adduct mimicking the tetrahedral intermediate. This suggested that Lys315 was somehow involved in accelerating the nucleophilic substitutions along the reaction pathway. The study was extended to modified substrates where the free carboxylate had been esterified. Neither acylation nor deacylation seemed severely impaired with these compounds, showing that the interaction between the enzyme and the substrate-free carboxylate did not play a major role in catalysis

The Importance of the Negative Charge of Beta-lactam Compounds in the Interactions With Active-site Serine Dd-peptidases and Beta-lactamases

The interaction between various penicillins and cephalosporins the carboxylate group of which at C-3 or C-4 had been esterified or amidated and different penicillin-recognizing enzymes was studied. In general, our findings reinforced the common assumption that an anionic group at that position is necessary for the effective acylation of these enzymes. However, the relative activities of the modified beta-lactams as inactivators of the Streptomyces R61 DD-peptidase or as substrates of the Bacillus licheniformis, Streptomyces albus G and Enterobacter cloacae beta-lactamases did not fit a general scheme in which the intrinsic electronic and geometric properties of the beta-lactam compounds would be sufficient to explain their substrate or inactivator properties towards the various types of enzymes investigated

Poly(3-alkylthiophene)s show unexpected second-order nonlinear optical response

Regioregular poly(3-hexylthiophene)s with chain lengths varying from 5 to 100 monomers are synthesized. Poly(3-hexylthiophene)s show in solution an unexpectedly significant second-order nonlinear optical response. The increase in transition dipole moment upon oligomerisation causes the significant second-order nonlinear optical response.crosscheck: This document is CrossCheck deposited related_data: Supplementary Information copyright_licence: The Royal Society of Chemistry has an exclusive publication licence for this journal copyright_licence: The accepted version of this article will be made freely available after a 12 month embargo period history: Received 22 October 2013; Accepted 16 January 2014; Accepted Manuscript published 16 January 2014; Advance Article published 30 January 2014; Version of Record published 13 February 2014status: publishe

The importance of the negative charge of beta-lactam compounds in the interactions with active-site serine DD-peptidases and beta-lactamases.

The interaction between various penicillins and cephalosporins the carboxylate group of which at C-3 or C-4 had been esterified or amidated and different penicillin-recognizing enzymes was studied. In general, our findings reinforced the common assumption that an anionic group at that position is necessary for the effective acylation of these enzymes. However, the relative activities of the modified beta-lactams as inactivators of the Streptomyces R61 DD-peptidase or as substrates of the Bacillus licheniformis, Streptomyces albus G and Enterobacter cloacae beta-lactamases did not fit a general scheme in which the intrinsic electronic and geometric properties of the beta-lactam compounds would be sufficient to explain their substrate or inactivator properties towards the various types of enzymes investigated

[Monnaie : Bronze, Édesse, Mésopotamie, Abgar]

Appartient à l’ensemble documentaire : MonnGr

Expression, purification, crystallization and preliminary X-ray analysis of the native class C beta-lactamase from Enterobacter cloacae 908R and two mutants.

Crystals have been obtained of the Enterobacter cloacae 908R beta-lactamase and two point mutants by the vapour-diffusion method using similar conditions [pH 9.0, polyethylene glycol (M(r) = 6000) as precipitant]. The three crystal forms belong to the orthorhombic space group P2(1)2(1)2, with roughly the same unit-cell parameters; i.e. for the wild-type crystals a = 46.46, b = 82.96, c = 95.31 A. In the best cases, the crystals diffract to about 2.1 A resolution on a rotating-anode X-ray source at room temperature. Co-crystallization experiments of poor substrates with the wild-type protein and the active-site serine mutant (S64C) are planned and should lead to a better understanding of the catalytic mechanism of class C beta-lactamases