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The Use of a β-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions
Published on: February 1, 2018
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Time-resolved β-lactam cleavage by L1 metallo-β-lactamase.
M Wilamowski1,2,3, D A Sherrell4, Y Kim1,4
1Center for Structural Genomics of Infectious Diseases, Consortium for Advanced Science and Engineering, University of Chicago, Chicago, IL, 60667, USA.
Nature Communications
|November 30, 2022
Summary
This study visualizes the moxalactam antibiotic binding and hydrolysis by L1 metallo-β-lactamase (MBL) using time-resolved crystallography. We captured key steps of the enzymatic reaction, revealing insights into antibiotic resistance mechanisms.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Metallo-β-lactamases (MBLs) are crucial in antibiotic resistance.
- Understanding MBL enzymatic mechanisms is vital for developing new therapeutics.
Purpose of the Study:
- To elucidate the structural dynamics of moxalactam hydrolysis by Stenotrophomonas maltophilia L1 MBL.
- To capture transient intermediates during the enzymatic reaction.
Main Methods:
- Time-resolved serial synchrotron X-ray crystallography at 2.20 Å resolution.
- UV-photolabile caged Zn²⁺ ions to initiate enzymatic activity.
- Time points captured from 20 ms to 4000 ms.
Main Results:
- Observed binding of two Zn²⁺ ions and moxalactam to the L1 MBL active site.
- Detected intact β-lactam ring for 100 ms, followed by cleavage at 150 ms.
- Conformational changes leading to a relaxed enzyme state by 2000 ms, with minimal shifts in metal ions and active site residues.
Conclusions:
- Provided a high-resolution, time-resolved view of β-lactam hydrolysis by L1 MBL.
- Mechanistic insights gained can inform the design of MBL inhibitors.
- The study offers a generalizable model for understanding other MBL enzymatic reactions.
