Mechanism of specific target recognition and RNA hydrolysis by ribonucleolytic toxin restrictocin.
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.
Nayak
S K
SK
Immunochemistry Laboratory, National Institute of Immunology, Aruna Asaf Ali Road, New Delhi 110067, India.
Bagga
S
S
Gaur
D
D
Nair
D T
DT
Salunke
D M
DM
Batra
J K
JK
D000485:Allergens; D000818:Animals; D052179:Antigens, Plant; D001120:Arginine / Q000737:chemistry / Q000235:genetics; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics; D006367:HeLa Cells; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009183:Mycotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000737:chemistry / Q000494:pharmacology; D012339:RNA, Ribosomal, 28S / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012156:Reticulocytes / Q000187:drug effects / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D013379:Substrate Specificity / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
C081947:ASPF1 protein, Aspergillus fumigatus; D000485:Allergens; D052179:Antigens, Plant; D005656:Fungal Proteins; D009183:Mycotoxins; D011500:Protein Synthesis Inhibitors; D012339:RNA, Ribosomal, 28S; D011994:Recombinant Proteins; C036196:MITF protein, Aspergillus restrictus; D018698:Glutamic Acid; D014443:Tyrosine; D006639:Histidine; D001120:Arginine; D012260:Ribonucleases