Unexpected advanced generation cephalosporinase activity of the M69F variant of SHV beta-lactamase
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The Use of a β-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions
Published on: February 1, 2018
Marion S Helfand1, Andrea M Hujer, Frank D Sönnichsen
1Infectious Disease Division, University Hospitals of Cleveland and Case Western Reserve University School of Medicine, Ohio 44106, USA.
Abstract:
Infections with bacteria that contain hydrolytic beta-lactamase enzymes are becoming a serious problem in the United States. Mutations at Met-69, an amino acid proximal to the active site Ser-70 in the TEM-1 and SHV-1 beta-lactamases, have emerged as a puzzling cause of bacterial resistance to inhibitors of beta-lactamases. Site-saturation mutagenesis of the 69 position in SHV beta-lactamase was performed to determine how mutations of this non-catalytic residue play a role in increasing 50% inhibitory concentrations (IC(50) concentrations) for clinically important beta-lactamase enzyme inhibitors. Two distinct phenotypes are evident in the variant beta-lactamases studied: significantly increased minimum inhibitory concentrations (microg/ml) and IC(50) concentrations to clavulanic acid for the Met69Ile, Leu, and Val substitutions, and unanticipated increased minimum inhibitory concentrations and hydrolytic activity toward ceftazidime, an advanced generation cephalosporin antibiotic, for the Met69Lys, Tyr- and Phe-substituted enzymes. Molecular modeling studies emphasize the conserved structure of these substitutions despite great variation in substrate specificity. This study demonstrates the key role of Met-69 in defining substrate specificity of SHV beta-lactamases and alerts us to new phenotypes that may emerge clinically.
Insights
Mutations in SHV beta-lactamase at Met-69 significantly alter bacterial resistance to antibiotics. This study reveals new resistance mechanisms against beta-lactamase inhibitors and advanced cephalosporins.
Area of Science:
- Microbiology
- Biochemistry
- Drug Discovery
Background:
- Bacterial infections with beta-lactamase enzymes pose a growing threat in the US.
- Mutations in beta-lactamases, particularly at Met-69, are linked to resistance against enzyme inhibitors.
- Understanding these mutations is crucial for developing effective treatments.
Purpose of the Study:
- To investigate the role of Met-69 mutations in SHV beta-lactamase.
- To determine how these mutations affect resistance to beta-lactamase inhibitors and antibiotics.
- To explore the impact on enzyme activity and substrate specificity.
Main Methods:
- Site-saturation mutagenesis of the Met-69 position in SHV beta-lactamase.
- Assays to measure minimum inhibitory concentrations (MICs) and 50% inhibitory concentrations (IC50s).
- Molecular modeling to analyze structural changes and substrate specificity.
Main Results:
- Met69Ile, Leu, and Val substitutions increased resistance to clavulanic acid.
- Met69Lys, Tyr, and Phe substitutions enhanced hydrolytic activity against ceftazidime.
- Molecular modeling showed conserved structures despite varied substrate specificity.
Conclusions:
- Met-69 is a key residue defining the substrate specificity of SHV beta-lactamases.
- Mutations at Met-69 can lead to novel resistance phenotypes.
- This research highlights emerging challenges in antibiotic resistance and treatment strategies.
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