Analysis of the context dependent sequence requirements of active site residues in the metallo-beta-lactamase IMP-1.

The metallo-beta-lactamase IMP-1 catalyzes the hydrolysis of a broad range of beta-lactam antibiotics to provide bacterial resistance to these compounds. In this study, 29 amino acid residue positions in and near the active-site pocket of the IMP-1 enzyme were randomized individually by site-directed mutagenesis of the corresponding codons in the bla(IMP-1) gene. The 29 random libraries were used to identify positions that are critical for the catalytic and substrate-specific properties of the IMP-1 enzyme. Mutants from each of the random libraries were selected for the ability to confer to Escherichia coli resistance to ampicillin, cefotaxime, imipenem or cephaloridine. The DNA sequence of several functional mutants was determined for each of the substrates. Comparison of the sequences of mutants obtained from the different antibiotic selections indicates the sequence requirements for each position in the context of each substrate. The zinc-chelating residues in the active site were found to be essential for hydrolysis of all antibiotics tested. Several positions, however, displayed context-dependent sequence requirements, in that they were essential for one substrate(s) but not others. The most striking examples included Lys69, Asp84, Lys224, Pro225, Gly232, Asn233, Asp236 and Ser262. In addition, comparison of the results for all 29 positions indicates that hydrolysis of imipenem, cephaloridine and ampicillin has stringent sequence requirements, while the requirements for hydrolysis of cefotaxime are more relaxed. This suggests that more information is required to specify active-site pockets that carry out imipenem, cephaloridine or ampicillin hydrolysis than one that catalyzes cefotaxime hydrolysis.

Molecular analysis of beta-lactamase structure and function.

The extensive and sometimes irresponsible use of beta-lactam antibiotics in clinical and agricultural settings has contributed to the emergence and widespread dissemination of antibiotic-resistant bacteria. Bacteria have evolved three strategies to escape the activity of beta-lactam antibiotics: 1) alteration of the target site (e.g. penicillin-binding protein (PBPs), 2) reduction of drug permeation across the bacterial membrane (e.g. efflux pumps) and 3) production of beta-lactamase enzymes. The beta-lactamase enzymes inactivate beta-lactam antibiotics by hydrolyzing the peptide bond of the characteristic four-membered beta-lactam ring rendering the antibiotic ineffective. The inactivation of the antibiotic provides resistance to the bacterium. Currently, there are over 300 beta-lactamase enzymes described for which numerous kinetic, structural, computational and mutagenesis studies have been performed. In this review, we discuss the recent work performed on the four different classes (A, B, C, and D) of beta-lactamases. These investigative advances further expand our knowledge about these complex enzymes, and hopefully, will provide us with additional tools to develop new inhibitors and antibiotics based on structural and rational designs.

Biochemical characterization of beta-lactamases Bla1 and Bla2 from Bacillus anthracis.

The Sterne and Ames strains of Bacillus anthracis carry chromosomal genes bla1 and bla2, which confer beta-lactam resistance when expressed in Escherichia coli. MIC measurements and steady-state kinetic analyses indicate that Bla1 possesses penicillinase activity while Bla2 possesses penicillinase, cephalosporinase, and carbapenem-hydrolyzing activities.