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The peptide toxins of poisonous Amanita mushrooms are bicyclic octapeptides (amatoxins) or heptapeptides (phallotox ins). In Amanita bisporigera, α-amanitin and phallacidin are synthesized as 35- and 34-amino acid proproteins, respectively, in which the amino acid sequences found in the mature toxins are flanked by conserved amino acid sequences. The presence of invariant Pro residues immediately upstream of the toxin regions and as the last predicted amino acid in the toxin regions themselves suggests that a Prospecific peptidase is responsible for the initial posttranslational processing of the Amanita toxin proproteins. We purified an enzyme from the phalloidin producing mushroom Conocybe albipes that cleaves a synthetic 22-mer phalloidin peptide to release the mature toxin peptide (AWLATCP). Mass spectrometric analysis of the purified pro tein combined with isolation and sequencing of the encoding gene indicates that the responsible processing enzyme is a member of the prolyl oligopeptidase (POP) subfamily of proteases (EC 3.4.21.26). The processing enzyme was able to use the chromogenic POP substrate benzyloxycarbonyl-Gly-Pro-p-nitro-anilide and was inhibited by the specific POP inhibitor benzyl oxycarbonyl-Pro-prolinal. Both Pro bonds in the pro-protein are cleaved by the same enzyme, with the Cterminal Pro bond cleaved first or much faster than the N-terminal Pro bond. Transient accumulation of the N-terminal intermediate indicates that cleavage is not strongly processive. A synthetic peptide rep resenting the phallacidin pro-protein was also cleaved by the POP of C. albipes, but a precursor of amanitin (which is not made by C. albipes) was cleaved inefficiently. [ABSTRACT FROM AUTHOR]