Mechanism of Specific Target Recognition and RNA Hydrolysis by Ribonucleolytic Toxin Restrictocin

Abstract

Restrictocin, a member of the fungal ribotoxin family, specifically cleaves a single phosphodiester bond in the 28S rRNA and potently inhibits eukaryotic protein synthesis. Residues Tyr47, His49, Glu95, Phe96, Pro97, Arg120, and His136 have been predicted to form the active site of restrictocin. In this study, we have individually mutated these amino acids to alanine to probe their role in restrictocin structure and function. The role of Tyr47, His49, Arg120, and His136 was further investigated by making additional mutants. Mutating Arg120 or His136 to alanine or the other amino acids rendered the toxin completely inactive, whereas mutating Glu95 to alanine only partially inactivated the toxin. Mutation of Phe96 and Pro97 to Ala had no effect on the activity of restrictocin. The Tyr47 to alanine mutant was inactive in inhibiting protein synthesis, and had a nonspecific ribonuclease activity on 28S rRNA similar to that shown previously for the His49 to Ala mutant. Unlike the His136 to Ala mutant, the double mutants containing Tyr47 or His49 mutated to alanine along with His136 did not compete with restrictocin to cause a significant reduction in the extent of cleavage of 28S rRNA. In a model of restrictocin and a 29-mer RNA substrate complex, residues Tyr47, His49, Glu95, Arg120, and His136 were found to be near the cleavage site on RNA. It is proposed that in restrictocin Glu95 and His136 are directly involved in catalysis, Arg120 is involved in the stabilization of the enzyme-substrate complex, Tyr47 provides structural stability to the active site, and His49 determines the substrate specificity.

Structural and Functional Studies of RegB, a New Member of a Family of Sequence-specific Ribonucleases Involved in mRNA Inactivation on the Ribosome

“…The E105A mutation abolishes RegB toxicity, and E19A and E19V lower it. Previous mutagenesis experiments with other ribonucleases (including YoeB) showed that mutants of the general base conserved residual activity (30,32,33 19 and His 48 C ␤ is slightly too long. This could be related to the observation that RegB is catalytically a "very poor" enzyme and could suggest that the formation of a productive enzyme-substrate complex involves, after a first rapid binding step, an infrequent rearrangement of the protein structure.…”

Fungal proteinaceous compounds with multiple biological activities

“…It recognized and cleaved a single phosphodiester bond specifically in a GAGA tetranucleotide located in a conserved stem and loop structure in ribosomal RNA (Nayak et al 2001). The active site of restrictocin for its ribonucleolytic activity is shown in Fig.…”

Ribotoxin restrictocin manifests anti-HIV-1 activity through its specific ribonuclease activity

“…The IC 50 of ␣-sarcin has been shown to be in the range of 0.4-10 M for different cell lines [6]. However, ribotoxins potently inhibit protein synthesis in permeabilized cells, and therefore are much more toxic to them [7][8][9]. Restrictocin is a non-glycosylated, basic, single chain protein of 149 amino acids with a molecular weight of 16.8 kDa [2,10].…”

“…They only cleave a single phosphodiester bond in 28s rRNA sparing all other cellular RNAs. The specific target recognition ability of restrictocin has been shown to be through a histidine residue at position 49, substitution of which results in the loss of specificity though the RNase activity of the protein is maintained [8,9]. The catalytic apparatus of restrictocin is similar to that of pyrimidine specific RNases of the RNase A superfamily, and histidine at position 136 has been shown to be absolutely essential for the ribonucleolytic activity of restrictocin [9].…”

“…The specific target recognition ability of restrictocin has been shown to be through a histidine residue at position 49, substitution of which results in the loss of specificity though the RNase activity of the protein is maintained [8,9]. The catalytic apparatus of restrictocin is similar to that of pyrimidine specific RNases of the RNase A superfamily, and histidine at position 136 has been shown to be absolutely essential for the ribonucleolytic activity of restrictocin [9]. Restrictocin being a cytotoxic molecule, has been used successfully in the construction of active immunotoxins for site specific delivery to cancer cells [11,12].…”