D-amino acids are present in some peptides from amphibian skin. These residues are derived from the corresponding L-amino acids present in the respective precursors. From skin secretions of Bombinae, we have isolated an enzyme that catalyzes the isomerization of an L-Ile in position 2 of a model peptide to D-allo-Ile. In the course of this reaction, which proceeds without the addition of a cofactor, radioactivity from tritiated water is incorporated into the second position of the product. The amino acid sequence of this isomerase could be deduced from cloned cDNA and genomic DNA. After expression of this cDNA in oocytes of Xenopus laevis, isomerase activity could be detected. Polypeptides related to the frog skin enzyme are present in several vertebrate species, including humans.amphibia ͉ bombinin H ͉ isomerase ͉ chirality F rom amphibian skin, a wide variety of biologically active peptides have been isolated (1). One of these compounds, the heptapeptide dermorphin (present in the skin of the South American tree frog Phyllomedusa sauvagei), is a potent opiate that binds with high affinity and selectivity to -opiate receptors (2). Quite unexpectedly, dermorphin was found to contain D-Ala at position 2, and this residue is essential for binding to the receptor. Subsequently, several additional opiate peptides containing a D-amino acid were found in the skin of other Phyllomedusinae (3). The bombinins H, antibacterial and hemolytic peptides isolated from skin secretions of the frog Bombina variegata, contain either L-Ile or D-allo-Ile (D-aIle) as the second amino acid (4). Peptides with D-amino acids in their sequences were also found in different invertebrate species, such as snails (5-7), the venom of a spider (8), and crustaceans (9). The most recent addition to this group was a homologue of C-type natriuretic peptides isolated from the venom of the male platypus (10).In principle, the biosynthesis of these D-amino acids in animal peptides has been clarified. As shown for dermorphin (11) and subsequently for several cases of vertebrate and invertebrate peptides, the secreted products are derived from larger precursors encoded by mRNAs. At the positions where a D-amino acid is present in the end product, a codon for the corresponding L-isomer has been found (reviewed in refs. 12 and 13). This finding implies that, at some stage in the processing of the precursors, a conversion of certain L-amino acids to the D-form takes place. Indeed, in many cases, the L and D forms of the mature peptides coexist in the respective secretory glands and secretions, which can be interpreted as incomplete isomerization. An enzyme that catalyzes such an unusual reaction has been characterized from the venom of a funnel web spider (8). In this case, the chirality of a Ser close to the C terminus of a peptide termed -agatoxin IV is inverted. This ''isomerase'' is related to Ser proteases and functions without any added cofactors (14, 15).As mentioned above, skin secretions of Bombina species contain a group of hemolytic peptides tha...