TY - JOUR
T1 - A continuous fluorometric assay for the assessment of MazF ribonuclease activity
AU - Wang, Nora R.
AU - Hergenrother, Paul J.
N1 - Funding Information:
This work was supported by the National Institutes of Health (R01-GM68385) and the Office of Naval Research (N00014-02-1-0390). N.R.W. was supported through the Department of Homeland Security (DHS) Scholarship and Fellowship Program.
PY - 2007/12/15
Y1 - 2007/12/15
N2 - 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 KM and Vmax 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.
AB - 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 KM and Vmax 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.
KW - Antibacterials
KW - Antibiotics
KW - High-throughput screening
KW - Protein-protein disruption
KW - Toxin-antitoxin systems
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U2 - 10.1016/j.ab.2007.07.017
DO - 10.1016/j.ab.2007.07.017
M3 - Article
C2 - 17706586
AN - SCOPUS:35549005884
SN - 0003-2697
VL - 371
SP - 173
EP - 183
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -