Open AccessLocalization of the catalytic activity in restrictocin molecule by deletion mutagenesis
Author(s) -
Nayak Surendra K.,
Batra Janendra K.
Publication year - 2000
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.2000.01176.x
Subject(s) - phosphodiester bond , amino acid , biochemistry , mutant , escherichia coli , biology , mutagenesis , site directed mutagenesis , chemistry , stereochemistry , microbiology and biotechnology , rna , gene
Restrictocin, produced by the fungus Aspergillus restrictus , is a highly specific ribonucleolytic toxin which cleaves a single phosphodiester bond between G4325 and A4326 in the 28S rRNA. It is a nonglycosylated, single‐chain, basic protein of 149 amino acids. The putative catalytic site of restrictocin includes Tyr47, His49, Glu95, Arg120 and His136. To map the catalytic activity in the restrictocin molecule, and to study the role of N‐ and C‐terminus in its activity, we have systematically deleted amino‐acid residues from both the termini. Three N‐terminal deletions removing 8, 15 and 30 amino acids, and three C‐terminal deletions lacking 4, 6, and 11 amino acids were constructed. The deletion mutants were expressed in Escherichia coli , purified to homogeneity and functionally characterized. Removal of eight N‐terminal or four C‐terminal amino acids rendered restrictocin partially inactive, whereas any further deletions from either end resulted in the complete inactivation of the toxin. The study demonstrates that intact N‐ and C‐termini are required for the optimum functional activity of restrictocin.