Substrate specificity of a peptidyl-aminoacyl-L/D-isomerase from frog skin

Abstract

In the skin of fire-bellied toads (Bombina species), an aminoacyl-L/D-isomerase activity is present which catalyses the post-translational isomerization of the L- to the D-form of the second residue of its substrate peptides. Previously, this new type of enzyme was studied in some detail and genes potentially coding for similar polypeptides were found to exist in several vertebrate species including man. Here, we present our studies to the substrate specificity of this isomerase using fluorescence-labeled variants of the natural substrate bombinin H with different amino acids at positions 1, 2 or 3. Surprisingly, this enzyme has a rather low selectivity for residues at position 2 where the change of chirality at the alpha-carbon takes place. In contrast, a hydrophobic amino acid at position 1 and a small one at position 3 of the substrate are essential. Interestingly, some peptides containing a Phe at position 3 also were substrates. Furthermore, we investigated the role of the amino-terminus for substrate recognition. In view of the rather broad specificity of the frog isomerase, we made a databank search for potential substrates of such an enzyme. Indeed, numerous peptides of amphibia and mammals were found which fulfill the requirements determined in this study. Expression of isomerases with similar characteristics in other species can therefore be expected to catalyze the formation of peptides containing D-amino acids.

Fig. 1

Isomerization reaction demonstrated for substrates with unnatural amino acids at the second position. RP–HPLC chromatograms of reaction mixtures of labeled (a) Ile-Phg-Gly and (b) Ile-Cs*-Gly model peptides after the indicated incubation time

Fig. 2

Isomerization reaction demonstrated for various substrates. RP–HPLC chromatograms of reaction mixtures of (a) a chimeric peptide with the amino terminal sequence Tyr-Met-Phe of a deltorphin and the rest from the standard peptide, (b) a truncated peptide derived from the β-defensin-like peptide; (c) B-chain of mammalian insulin and (d) neuropeptide FF. In panels C,D peptides without a fluorescent marker (200 μM each) were used

Fig. 3

The isomerization reaction proceeds in both directions. RP–HPLC chromatograms of reaction mixtures of (a) Ile-Phe-Gly peptide or (b) Ile-(D-Phe)-Gly peptide are shown (arrows). Peptides without a fluorescent marker (250 μM each) were used