Role of histidine-50, glutamic acid-96, and histidine-137 in the ribonucleolytic mechanism of the ribotoxin ?-sarcin

Abstract

alpha-Sarcin is a ribotoxin secreted by the mold Aspergillus giganteus that degrades the ribosomal RNA by acting as a cyclizing ribonuclease. Three residues potentially involved in the mechanism of catalysis--histidine-50, glutamic acid-96, and histidine-137--were changed to glutamine. Three different single mutation variants (H50Q, E96Q, H137Q) as well as a double variant (H50/137Q) and a triple variant (H50/137Q/E96Q) were prepared and isolated to homogeneity. These variants were spectroscopically (circular dichroism, fluorescence emission, and proton nuclear magnetic resonance) characterized. According to these results, the three-dimensional structure of these variants of alpha-sarcin was preserved; only very minor local changes were detected. All the variants were inactive when assayed against either intact ribosomes or poly(A). The effect of pH on the ribonucleolytic activity of alpha-sarcin was evaluated against the ApA dinucleotide. This assay revealed that only the H50Q variant still retained its ability to cleave a phosphodiester bond, but it did so to a lesser extent than did wild-type alpha-sarcin. The results obtained are interpreted in terms of His137 and Glu96 as essential residues for the catalytic activity of alpha-sarcin (His137 as the general acid and Glu96 as the general base) and His50 stabilizing the transition state of the reaction catalyzed by alpha-sarcin.

Fungal extracellular ribotoxins as insecticidal agents

“…E. coli BL21 (DE3) cells, previously cotransformed with a thioredoxin-producing plasmid (pT-Trx) and the corresponding plasmid (pINPGαSH137Q), were used to produce the catalytically inactive -sarcin H137Q mutant, also as previously described (García-Ortega et al, 2000;Lacadena et al, 1994Lacadena et al, , 1995Lacadena et al, , 1999. This mutant retains the structural features of the wild-type protein, as well as its ability to interact with membranes, but lacks the characteristic ribonucleolytic activity of ribotoxins (Lacadena et al, 1995(Lacadena et al, , 1999. SDS-PAGE of proteins, Western blots, protein hydrolysis, amino acid analysis, and spectroscopic characterization were performed according to standardized procedures described before (García-Ortega et al, 2000, 2002Lacadena et al, 1994;Martínez-Ruiz et al, 2001).…”

Antifungal proteins: targets, mechanisms and prospective applications

“…It is a type 1 RIP and specifically cleaves one phosphodiester bond of the 28S rRNA [139]. The protein has been reported to act as a cyclising ribonuclease [140], and moreover, essential amino acids of the catalytic core have been identified by several mutational approaches [141,142]. a-Sarcin interacts with negatively charged model membranes [143,144], and a nineamino-acid peptide fragment of a-sarcin has been reported to be a membrane-perturbing structure [145].…”

Production and characterization of a noncytotoxic deletion variant of the Aspergillus fumigatus allergen Aspf1 displaying reduced IgE binding

“…The specific ribonucleolytic activity of ribotoxins was followed by detecting the release of the 400-nt a-fragment [2] from a cell-free reticulocyte lysate (Promega Corporation, Madison, WI, USA) when protein amounts were added in the 50-200 ng range [16,23,27]. The production of this 400-nt a-fragment was visualized by ethidium bromide staining after electrophoresis on 2.4% (w ⁄ v) agarose gels.…”

“…The activity of the purified proteins against poly(A) was assayed at pH 7.0 in 15% (w ⁄ v) polyacrylamide gels containing 0.1% (w ⁄ v) SDS and 0.3 mgAEmL )1 homopolynucleotide. In these zymograms, proteins exhibiting ribonuclease activity appear as colorless bands after appropriate destaining [2,16,27]. Volumograms of these bands, obtained with a photo documentation system UVI-Tec and the software facility UVIsoft UVI band Windows Application V97.04, were used to quantify the activity.…”