The cyclic peptide toxins of deadly Amanita mushrooms represent a group of natural products with unique structures and potent bioactivities. Amatoxins such as α-amanitin and the phallotoxins such as phalloidin are bicyclic peptides whose propeptides are synthesized on ribosomes (i.e., they are “RiPPs”). In addition to the genes for amatoxins and phallotoxins, the genomes of A. bisporigera and A. phalloides contain more than 40 related sequences, called the “MSDIN” gene family, with the potential to make dozens of previously undescribed cyclic peptides. The MSDIN family is characterized by conserved N and C terminal sequences flanking a hypervariable region from which the mature toxins are derived. The hypervariable region is flanked by invariant Pro residues. The 35-amino acid α-amanitin propeptide is processed by a specialized prolyl oligopeptidase (POPB) in the amanitin-producing mushroom Galerina marginata. POPB cuts the propeptide at the two flanking Pro residues and cyclizes the resulting octapeptide to cyclo(IWGIGCNP). The two reactions catalyzed by POPB (a hydrolysis and a transpeptidation) are nonprocessive, the intermediate being the C-terminal 25mer. POPB has a Km of 25 μM and a turnover rate of 6 sec-1, comparable in efficiency to other known peptide macrocyclases. POPB requires an α-helix secondary structure in the C-terminal half of the propeptide. It can cyclize many sequence variants of amanitin, and based on the cyclic peptides that Amanita mushrooms are known to make, it is predicted to cyclize peptides of 6 to 10 amino acids.