Localization of the catalytic activity in restrictocin molecule by deletion mutagenesis.
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.
Nayak
S K
SK
Immunochemistry Laboratory, National Institute of Immunology, New Delhi, India.
Shveta
Batra
J K
JK
D000485:Allergens; D000596:Amino Acids / Q000737:chemistry; D052179:Antigens, Plant; D001230:Aspergillus / Q000378:metabolism*; D002384:Catalysis; D002942:Circular Dichroism; D004926:Escherichia coli; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009183:Mycotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology; D012260:Ribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D012270:Ribosomes / Q000187:drug effects; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
C081947:ASPF1 protein, Aspergillus fumigatus; D000485:Allergens; D000596:Amino Acids; D052179:Antigens, Plant; D005656:Fungal Proteins; D009183:Mycotoxins; D011993:Recombinant Fusion Proteins; C036196:MITF protein, Aspergillus restrictus; D012260:Ribonucleases