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Updated: Aug 19, 2026
Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance
Published on: May 2, 2018
Impact of remote mutations on metallo-beta-lactamase substrate specificity: implications for the evolution of
Peter Oelschlaeger1, Stephen L Mayo, Juergen Pleiss
1Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
Abstract:
Metallo-beta-lactamases have raised concerns due to their ability to hydrolyze a broad spectrum of beta-lactam antibiotics. The G262S point mutation distinguishing the metallo-beta-lactamase IMP-1 from IMP-6 has no effect on the hydrolysis of the drugs cephalothin and cefotaxime, but significantly improves catalytic efficiency toward cephaloridine, ceftazidime, benzylpenicillin, ampicillin, and imipenem. This change in specificity occurs even though residue 262 is remote from the active site. We investigated the substrate specificities of five other point mutants resulting from single-nucleotide substitutions at positions near residue 262: G262A, G262V, S121G, F218Y, and F218I. The results suggest two types of substrates: type I (nitrocefin, cephalothin, and cefotaxime), which are converted equally well by IMP-6, IMP-1, and G262A, but even more efficiently by the other mutants, and type II (ceftazidime, benzylpenicillin, ampicillin, and imipenem), which are hydrolyzed much less efficiently by all the mutants. G262V, S121G, F218Y, and F218I improve conversion of type I substrates, whereas G262A and IMP-1 improve conversion of type II substrates, indicating two distinct evolutionary adaptations from IMP-6. Substrate structure may explain the catalytic efficiencies observed. Type I substrates have R2 electron donors, which may stabilize the substrate intermediate in the binding pocket. In contrast, the absence of these stabilizing interactions with type II substrates may result in poor conversion. This observation may assist future drug design. As the G262A and F218Y mutants confer effective resistance to Escherichia coli BL21(DE3) cells (high minimal inhibitory concentrations), they are likely to evolve naturally.
Insights
Metallo-beta-lactamase mutations alter antibiotic resistance. Specific mutations enhance the breakdown of certain antibiotics, suggesting potential evolutionary pathways for drug resistance in bacteria.
Area of Science:
- Microbiology
- Biochemistry
- Drug Discovery
Background:
- Metallo-beta-lactamases (MBLs) pose a significant threat due to their broad-spectrum hydrolysis of beta-lactam antibiotics.
- The G262S mutation in IMP-1, compared to IMP-6, alters substrate specificity, enhancing catalytic efficiency against several key antibiotics.
Purpose of the Study:
- To investigate the impact of single-nucleotide substitutions near residue 262 on the substrate specificity of metallo-beta-lactamase IMP variants.
- To understand the structural basis for altered catalytic efficiency and substrate preference in MBLs.
Main Methods:
- Site-directed mutagenesis was used to create five point mutants (G262A, G262V, S121G, F218Y, F218I).
- Enzyme kinetics were analyzed to determine the catalytic efficiency of wild-type and mutant MBLs against various beta-lactam substrates.
- Minimal inhibitory concentrations (MICs) were determined for resistant bacterial strains.
Main Results:
- Mutants exhibited differential substrate specificities, categorizing substrates into Type I (nitrocefin, cephalothin, cefotaxime) and Type II (ceftazidime, benzylpenicillin, ampicillin, imipenem).
- Mutants G262V, S121G, F218Y, and F218I enhanced Type I substrate conversion, while G262A and IMP-1 favored Type II substrate conversion.
- The G262A and F218Y mutants conferred high-level resistance in Escherichia coli, indicating potential for natural evolution.
Conclusions:
- Single point mutations can significantly alter MBL substrate specificity, leading to distinct evolutionary adaptations.
- Substrate structure, particularly the presence of R2 electron donors, influences catalytic efficiency by stabilizing intermediates.
- Understanding these structure-activity relationships can inform future antibiotic drug design and resistance management strategies.
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