Antonio Martı́nez-Ruiz

Ribotoxins are a more widespread group of proteins within the filamentous fungi than previously believed.

Alpha-sarcin, restrictocin and mitogillin are the best known members of the family of fungal ribotoxins. In recent years, new members of this family have been discovered and characterised. In this work, we study the occurrence of ribotoxins among different species of fungi. The presence of ribotoxins has been identified in some new species by means of genetic studies, as well as expression and activity assays. The ribotoxin genes have been partially sequenced, and demonstrate a high degree of similarity. These studies demonstrate that these toxins are more widespread than previously considered. This is surprising, considering the ribotoxins are such specific and potent toxins, of unknown biological function. These studies confirm the hypothesis that these proteins are naturally engineered toxins derived from ribonucleases of broad substrate specificity.

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

α‐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 dif‐ ferent 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 α‐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 α‐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 α‐sarcin. The results obtained are interpreted in terms of His137 and Glu96 as essential residues for the catalytic activity of α‐sarcin (His137 as the general acid and Glu96 as the general base) and His50 stabilizing the transition state of the reaction catalyzed by α‐sarcin. Proteins 1999;37:474–484. ©1999 Wiley‐Liss, Inc.

The cytotoxin α‐sarcin behaves as a cyclizing ribonuclease

The hydrolysis of adenylyl(3′→5′)adenosine (ApA) and guanylyl(3′→5′)adenosine (GpA) dinucleotides by the cytotoxic protein α‐sarcin has been studied. Quantitative analysis of the reaction has been performed through reverse‐phase chromatographic (HPLC) separation of the resulting products. The hydrolysis of the 3′‐5′ phosphodiester bond of these substrates yields the 2′‐3′ cyclic mononucleotide; this intermediate is converted into the corresponding 3′‐monophosphate derivative as the final product of the reaction. The values of the apparent Michaelis constant (K M), k cat and k cat/K M have also been calculated. The obtained results fit into a two‐step mechanism for the enzymatic activity of α‐sarcin and allow to consider this protein as a cyclizing RNase.

Characterization of a natural larger form of the antifungal protein (AFP) from Aspergillus giganteus

Two major proteins, alpha-sarcin and an antifungal polypeptide (AFP), are secreted by the mould Aspergillus giganteus MDH 18894 when it is cultured for 70-80 h. A third major protein is also found in the extracellular medium at 48-60 h, but it disappears as the culture proceeds. This protein has been isolated and characterized in terms of apparent molecular mass, electrophoretic and chromatographic behaviour, NH2-terminal primary structure, amino acid content, spectroscopical features, reactivity against anti-AFP antibodies, and antifungal activity. Based on the obtained results it would be an extracellular inactive precursor form of AFP, designated as the large form of AFP (lf-AFP). Its amino acid composition is identical to that of AFP but containing six extra residues. NH2-terminal sequence analysis of the first eight amino acid residues of this polypeptide revealed that the extra residues can be perfectly accommodated within the DNA-deduced sequence of the precursor form of AFP. Its alignment with precursor sequences of different proteins, secreted by a variety of Aspergillus spp., reveals the existence of a common tetrapeptide at the carboxy-terminal end of their leader peptides. This sequence would be Ile/Leu-Xaa-Yaa-Arg, being mostly Xaa and Yaa an acid residue (Asp/Glu) and alanine, respectively. The presence of lf-AFP as an extracellular protein would be in perfect agreement with the existence of this tetrapeptide motif, that can be involved in the protein secretion mechanisms of filamentous fungi.

The solubility of the ribotoxin alpha-sarcin, produced as a recombinant protein in Escherichia coli, is increased in the presence of thioredoxin.

The yield of purified recombinant α‐sarcin increases approximately three‐ to fourfold when this toxin is co‐expressed in Escherichia coli with thioredoxin. This increased production is attributed to the existence, in the presence of thioredoxin, of a reducing environment which allows rearrangement of incorrect disulphide bonds to produce the soluble native conformation. The protein thus produced retains the structural, spectroscopic and enzymatic features of the natural fungal α‐sarcin.