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Single amino acid substitutions affecting the specificity of the fungal ribotoxin mitogillin

R Kao1, J Davies

  • 1Department of Microbiology and Immunology, The University of British Columbia, 6174 University Blvd., Vancouver, B.C., Canada. kao@davies.microbiology.ubc.ca

FEBS Letters
|January 29, 2000
PubMed

Insights

Mitogillin, a fungal ribotoxin, inhibits protein synthesis by cleaving ribosomal RNA. Mutations in specific domains altered its ribonuclease activity and target specificity, confirming their roles in RNA binding and cleavage.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Mycology

Background:

  • Mitogillin is a fungal ribotoxin that inhibits protein synthesis.
  • It functions by cleaving the alpha-sarcin/ricin loop (SRL) in ribosomal RNA.
  • Mitogillin is derived from a T1/U2-like ribonuclease with added domains for target specificity.

Purpose of the Study:

  • To investigate the functional roles of specific domains in mitogillin.
  • To determine how amino acid substitutions affect mitogillin's ribonuclease activity and substrate specificity.

Main Methods:

  • Site-directed mutagenesis was used to alter amino acids in the B1-L1-B2 and L4 regions of mitogillin.
  • Mutant activities were assessed using polyinosinic acid and specific cleavage of the SRL.

Main Results:

  • Asn7Ala and Lys111Gln substitutions resulted in altered ribonuclease activity.
  • These mutations also led to diminished substrate specificity for SRL cleavage.
  • The findings support the proposed functions of the targeted domains in mitogillin's activity.

Conclusions:

  • The B1-L1-B2 and L4 domains are critical for mitogillin's ribonuclease activity and specificity.
  • Amino acid residues Asn7 and Lys111 play key roles in these functions.

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