Jk. Nayak, Sk",shveta,"batra, Localization of the catalytic activity in restrictocin molecule by deletion mutagenesis, EUR J BIOCH, 267(6), 2000, pp. 1777-1783
Restrictocin, produced by the fungus Aspergillus restrictus, is a highly sp
ecific ribonucleolytic toxin which cleaves a single phosphodiester bond bet
ween G4325 and A4326 in the 28S rRNA. It is a nonglycosylated, single-chain
, basic protein of 149 amino acids. The putative catalytic site of restrict
ocin 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-te
rminus in its activity, we have systematically deleted amino-acid residues
from both the termini. Three N-terminal deletions removing 8, 15 and 30 ami
no acids, and three C-terminal deletions lacking 4, 6, and 11 amino acids w
ere constructed. The deletion mutants were expressed in Escherichia coli, p
urified to homogeneity and functionally characterized. Removal of eight N-t
erminal or four C-terminal amino acids rendered restrictocin partially inac
tive, whereas any further deletions from either end resulted in the complet
e inactivation of the toxin. The study demonstrates that intact N- and C-te
rmini are required for the optimum functional activity of restrictocin.