Related Experiment Videos
Probing the active site of mitogillin, a fungal ribotoxin
1Department of Microbiology and Immunology, The University of British Columbia, Vancouver, Canada. kao@davies.microbiology.ubc.ca
Abstract:
Fungal ribotoxins, such as mitogillin and the related Aspergillus toxins restrictocin and alpha-sarcin, are highly specific ribonucleases, which inactivate the ribosome enzymatically by cleaving the eukaryotic 28S RNA of the large ribosomal subunit at a single phosphodiester bond. The site of cleavage occurs between G4325 and A4326, which are present in a 14-base sequence (the alpha-sarcin loop) conserved among the large subunit rRNAs of all living species. The amino acid residues involved in the cytotoxic activities of mitogillin were investigated by introducing point mutations using hydroxylamine into a recombinant Met-mature mitogillin (mitogillin with a Met codon at the N-terminus and no leader sequence) gene constructed from an Aspergillus fumigatus cDNA clone. These constructs were cloned into a yeast expression vector under the control of the GAL1 promoter and transformed into Saccharomyces cerevisiae. Upon induction of mitogillin expression, surviving transformants revealed that substitutions of certain amino acid residues on mitogillin abolished its cytotoxicity. Non-toxic mutant genes were cloned into an Escherichia coli expression vector, the proteins overexpressed and purified to homogeneity and their activities examined by in vitro ribonucleolytic assays. These studies identified the His-49Tyr, Glu-95Lys, Arg-120Lys and His-136Tyr mutations to have a profound impact on the ribonucleolytic activities of mitogillin. We conclude that these residues are key components of the active site contributing to the catalytic activities of mitogillin.
Insights
Fungal ribotoxins like mitogillin are specific ribonucleases that inactivate ribosomes. Key amino acid residues, including His-49, Glu-95, Arg-120, and His-136, are crucial for mitogillin
Area of Science:
- Biochemistry
- Molecular Biology
- Mycology
Background:
- Fungal ribotoxins, such as mitogillin, alpha-sarcin, and restrictocin, are potent ribonucleases.
- These toxins target the 28S ribosomal RNA (rRNA) of the large ribosomal subunit, cleaving it at a conserved G4325-A4326 phosphodiester bond within the alpha-sarcin loop.
- This enzymatic activity leads to the inactivation of the ribosome and cellular toxicity.
Purpose of the Study:
- To identify the specific amino acid residues within mitogillin essential for its cytotoxic and ribonucleolytic activities.
- To elucidate the role of these residues in the catalytic mechanism of fungal ribotoxins.
Main Methods:
- Site-directed mutagenesis using hydroxylamine was employed to generate mutant mitogillin genes.
- Recombinant mitogillin variants were expressed in Saccharomyces cerevisiae and Escherichia coli.
- Purified mutant proteins were subjected to in vitro ribonucleolytic assays to assess their enzymatic activity.
Main Results:
- Mutations at specific amino acid positions, namely His-49Tyr, Glu-95Lys, Arg-120Lys, and His-136Tyr, significantly abolished mitogillin's cytotoxicity.
- These identified mutations demonstrated a profound impact on the in vitro ribonucleolytic activity of mitogillin.
- The study successfully pinpointed critical residues involved in the catalytic function.
Conclusions:
- The amino acid residues His-49, Glu-95, Arg-120, and His-136 are indispensable components of the mitogillin active site.
- These residues play a critical role in the catalytic activity and cytotoxic effects of fungal ribotoxins.
- Understanding these key residues advances knowledge of ribotoxin mechanisms and potential therapeutic applications.