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Novel metallo beta-lactamase mediated by a Shigella flexneri plasmid
1Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan. oharak@p.chiba-u.ac.jp
Abstract:
Novel carbapenem-hydrolyzing beta-lactamase (newly named MET-1) encoded on a transferable plasmid pMS390 from Shigella flexneri JS19622 was purified. The molecular weight was 28,000 by SDS-PAGE and the isoelectric point was higher than 9.3. This beta-lactamase favorably hydrolyzed classical cephalosporins and oxyimino-cephalosporins rather than penicillins and carbapenems, but did not hydrolyze monobactams. The enzymatic activity was inhibited by EDTA, and the enzyme was found to contain two moles of zinc per mole of enzyme protein by means of atomic absorption spectrophotometry. These results indicated that the enzyme is a zinc beta-lactamase which differs from known metallo beta-lactamases, especially in its cephalosporinase-type substrate profile.
Insights
A novel zinc beta-lactamase, MET-1, was identified in Shigella flexneri. This enzyme hydrolyzes cephalosporins but not carbapenems, distinguishing it from other metallo-beta-lactamases.
Area of Science:
- Microbiology
- Enzymology
- Biochemistry
Background:
- Beta-lactamase enzymes confer bacterial resistance to beta-lactam antibiotics.
- Carbapenem-resistant Enterobacteriaceae pose a significant global health threat.
- Novel beta-lactamases require characterization to understand resistance mechanisms.
Purpose of the Study:
- To purify and characterize a novel carbapenem-hydrolyzing beta-lactamase (MET-1).
- To determine the substrate profile and biochemical properties of MET-1.
- To differentiate MET-1 from previously identified metallo-beta-lactamases.
Main Methods:
- Purification of MET-1 from Shigella flexneri JS19622.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for molecular weight determination.
- Enzyme kinetics assays to determine substrate hydrolysis profile.
- Atomic absorption spectrophotometry for metal content analysis.
Main Results:
- MET-1 was purified with a molecular weight of 28,000 Da and an isoelectric point >9.3.
- The enzyme efficiently hydrolyzed cephalosporins and oxyimino-cephalosporins but not penicillins or carbapenems.
- Enzymatic activity was inhibited by EDTA, and the enzyme contained zinc, indicating it is a zinc beta-lactamase.
- MET-1 exhibited a cephalosporinase-type substrate profile, distinct from known metallo-beta-lactamases.
Conclusions:
- MET-1 is a novel zinc-dependent beta-lactamase with a unique substrate specificity.
- Its plasmid-encoded nature suggests potential for horizontal gene transfer and spread of resistance.
- Understanding MET-1's characteristics is crucial for developing strategies against carbapenemase-producing bacteria.