A continuous fluorometric assay for the assessment of MazF ribonuclease activity
Nora R Wang1, Paul J Hergenrother
1Department of Chemistry, Roger Adams Laboratory, University of Illinois, Urbana, IL 61801, USA.
Abstract:
Plasmids maintain themselves in their bacterial host through several different mechanisms, one of which involves the synthesis of plasmid-encoded toxin and antitoxin proteins. When the plasmid is present, the antitoxin binds to and neutralizes the toxin. If a plasmid-free daughter cell arises, however, the labile antitoxin is degraded (and not replenished) and the toxin kills the cell from within. These toxin-antitoxin (TA) systems thereby function as postsegregational killing systems, and the disruption of the TA interaction represents an intriguing antibacterial strategy. It was recently discovered that the genes for one particular TA system, MazEF, are ubiquitous on plasmids isolated from clinical vancomycin-resistant enterococci (VRE) strains. Thus, it appears that small molecule disruptors of the MazEF interaction have potential as antibacterial agents. The MazF toxin protein is known to be a ribonuclease. Unfortunately, traditional methods for the assessment of MazF activity rely on the use of radiolabeled substrates followed by analysis with polyacrylamide gel electrophoresis. This article describes a simple and convenient continuous assay for the assessment of MazF activity. The assay uses an oligonucleotide with a fluorophore on the 5' end and a quencher on the 3' end, and processing of this substrate by MazF results in a large increase in the fluorescence signal. Through this assay, we have for the first time determined K(M) and V(max) values for this enzyme and have also found that MazF is not inhibited by standard ribonuclease inhibitors. This assay will be useful to those interested in the biochemistry of the MazF family of toxins and the disruption of MazE/MazF.
Insights
Toxin-antitoxin systems like MazEF are crucial for plasmid maintenance in bacteria. A new continuous assay allows for efficient assessment of MazF ribonuclease activity, aiding the development of novel antibacterial agents targeting VRE.
Area of Science:
- Bacteriology
- Molecular Biology
- Biochemistry
Background:
- Plasmids utilize toxin-antitoxin (TA) systems for stable inheritance in bacterial hosts.
- The MazEF TA system is prevalent in clinical vancomycin-resistant enterococci (VRE) plasmids.
- Disrupting TA interactions presents a promising antibacterial strategy.
Purpose of the Study:
- To develop a simple and continuous assay for assessing MazF toxin activity.
- To characterize the enzymatic properties of MazF, including kinetic parameters.
- To evaluate the potential of MazF as a target for novel antibacterial agents.
Main Methods:
- A novel continuous assay utilizing a fluorophore/quencher-labeled oligonucleotide substrate was developed.
- MazF ribonuclease activity was measured by the increase in fluorescence upon substrate cleavage.
- Enzyme kinetics (K(M), V(max)) were determined, and inhibition by standard RNase inhibitors was assessed.
Main Results:
- A convenient continuous assay for MazF activity was established.
- For the first time, K(M) and V(max) values for MazF were determined.
- MazF was found to be resistant to standard ribonuclease inhibitors.
Conclusions:
- The developed assay provides a valuable tool for studying MazF biochemistry.
- Understanding MazF activity is crucial for developing antibacterial strategies targeting VRE.
- The MazEF system, particularly MazF, represents a potential target for novel therapeutics against resistant bacteria.
