Didier Monnaie

Kinetic study of interaction between BRL 42715, beta-lactamases, and D-alanyl-D-alanine peptidases.

A detailed kinetic study of the interactions between BRL 42715, a beta-lactamase-inhibiting penem, and various beta-lactamases (EC 3.5.2.6) and D-alanyl-D-alanine peptidases (DD-peptidases, EC 3.4.16.4) is presented. The compound was a very efficient inactivator of all active-site serine beta-lactamases but was hydrolyzed by the class B, Zn(2+)-containing enzymes, with very different kcat values. Inactivation of the Streptomyces sp. strain R61 extracellular DD-peptidase was not observed, and the Actinomadura sp. strain R39 DD-peptidase exhibited a low level of sensitivity to the compound.

Interaction of clavulanate with class C β-lactamases

The interactions between clavulanate and three class C enzymes have been studied in detail. In all cases, the reactions followed branched pathways where 25–150 turnovers occurred before inactivation was completed. Reactivation rates were quite low. The poor efficiency of clavulanate as a class C inactivator appeared to rest upon a very slow acylation of the protein, and of a relatively high turnover rate.

A rapid-kinetic study of the class C β-lactamase of Enterobacter cloacae 908R

The individual rate constants for acylation and deacylation (k 2 and k 3, respectively) of the class C β‐lactamase of Enterobacter cloacae 908R by ampicillin and carbenicillin have been determined. For several other β‐lactams, the value of k 2 was too high to be determined and the k 2/k 3 ratio could be larger than 10,000. Branched pathways were also shown to occur with several penicillins and cephalosporins.

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.

Mechanism of action of β-lactamases and DD-peptidases

The introduction of penicillins as antibacterial agents probably represents one of the major breakthroughs of chemotherapy during the present century. However, the bacterial world did not remain without reaction and resistance to strains started to appear almost as soon as penicillin utilisation became popular. This resulted in an endless race between the chemists and the microbiologists on one side and the bacteria on the other, the formers discovering and synthesizing new compounds while the latters found new tricks to escape their lethal action so that the s-lactam family now comprises molecules of widely different structures of which the s-lactam ring remains the only common feature (Figure 1A).