A novel isomerase from amphibien skin

Skin of different amphibia synthesize peptides where the second amino acid is in the D-configuration. These peptides are derived from precursors which contain only L-amino acids. We have searched for an enzyme which could catalyze in these peptides the formation of D-amino acids from the corresponding L-isomers. From skin secretions of two Bombina species, we have isolated a novel enzyme which catalyzes the conversion of L- isoleucine, the second amino acid in a model peptide, to D-allo-isoleucine. This enzyme, which could be called a peptidyl-aminoacyl-L-D-isomerase, has been purified close to homogeneity. Within the near future, we hope to be able to determine parts of the amino acid sequence of this protein. Using this information, we want to clone from Bombina skin mRNA the cDNA coding for this enzyme. This cDNA will then be expressed with recombinant vaccinia virus to obtain sufficient quantitites of enzyme protein for studies on substrate specificity, reaction mechanism, etc. of this unusual reaction. We also want to search for related enzymes in skin of other frog species, in tissues other than skin, and for their possible presence in mammals. In a side fraction obtained during the purification of the isomerase from Bombina skin secretions, a small protein was detected which was termed Bv8. This contains 77 amino acids and is related to protein A, a non-toxic constituent of the venom of the black mamba. An EST from mouse embryonic stem cells has been deposited in the data banks which apparently codes for a fragment of a similar peptide. As a second project, we want to clone cDNAs coding for the precursor of Bv8 and its putative mouse homologue and also express these in animal cells. We will then also search for the presence of this protein in other species and tissues. From the side fraction mentioned above we will isolate milligram quantities of Bv8 which will then be used for diverse pharmacological in vivo and in vitro tests to search for a possible biological function.