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.2005 Mar 22;102(12):4235-9.
doi: 10.1073/pnas.0500789102. Epub 2005 Mar 9.

Biosynthesis of a D-amino acid in peptide linkage by an enzyme from frog skin secretions

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Biosynthesis of a D-amino acid in peptide linkage by an enzyme from frog skin secretions

Alexander Jilek et al. Proc Natl Acad Sci U S A..

Abstract

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.

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Figures

Fig. 1.
Fig. 1.
Characterization of the reaction product. (A) Separation of substrate and product by reverse-phase HPLC on an Aquapore RP300 column (see ref. for details). Substrate (15 μg in 50 μl of buffer) and ≈1 μg of crude enzyme were incubated for 8 h at 37°C. The arrow indicates the position in the gradient where synthetic Ile-d-aIle-Gly-Pro-Val-Leu-Gly-Ser.amide is eluted from the column (at ≈25.9% acetonitrile). Product yield was ≈8%. (B) Substrate (15 μg) and enzyme were incubated in 50 μl of tritiated water (specific activity, 5 Ci/ml (1 Ci = 37 GBq); American Radiolabeled Chemicals, St. Louis) for 8 h at 37°C: Subsequently, the sample was dried, redissolved several times in water, and dried again. Manual Edman degradation was performed by using standard conditions. After each step, radioactivity was extracted inton-butyl acetate and measured in a scintillation counter. Radioactivity (counts per min × 10–3) is plotted against the step of Edman degradation. In the control incubated in the absence of enzyme, <300 counts per min could be extracted after each Edman cycle.
Fig. 2.
Fig. 2.
Time course of the conversion of the peptide Ile-Ile-Gly-Pro-Val-Leu-Gly-Cys-amide, labeled with a fluorophor (seeMaterials and Methods), to the product containingd-aIle as the second residue. Reaction conditions were 100 mM phosphate buffer (pH 6.5) and 5 mM EDTA (•). In a parallel sample, the enzyme was preincubated in the presence of 1 mM diethyl pyrocarbonate for 16 h on ice (▪). Aliquots removed at different times were analyzed by reverse-phase HPLC.
Fig. 3.
Fig. 3.
Isomerase activity at different pH values. The same substrate as described in the legend to Fig. 2 was used. Relative activity is plotted against the pH.
Fig. 4.
Fig. 4.
Amino acid sequence deduced from cloned genomic DNA fromB. orientalis (21). Amino acid sequences obtained from the isomerase purified from skin secretions ofB.variegata are underlined. The singleN-glycosylation site is indicated (+).
Fig. 5.
Fig. 5.
Expression of isomerase cRNA inXenopus oocytes. Product yield in pmol is plotted against the number of injected oocytes from which supernatants were collected (▪). As controls, supernatants from mock-injected oocytes were analyzed in parallel (•).
Fig. 6.
Fig. 6.
Comparison of amino acid sequences of theBombina skin isomerase with other vertebrate sequences deduced from cDNA or genomic DNA. Man, domain H of the human IgG-Fc binding protein (25); Bird, chicken EST BM488067; Fish, FuguGenscan_28655. Asterisks indicate conserved His and Cys residues.
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References

    1. Lazarus, L. H. & Attila, M. (1993) Prog. Neurobiol. 41, 473–507. - PubMed
    1. Montecucchi, P. C., de Castiglione, R., Piani, S., Gozzini, L. & Erspamer, V. (1981) Int. J. Pept. Protein Res. 17, 275–283. - PubMed
    1. Erspamer, V., Melchiorri, P., Falconieri Erspamer, G., Negri, L., Corsi, R., Severini, C., Barra, D., Simmaco, M. & Kreil, G. (1989) Proc. Natl. Acad. Sci. USA 86, 5188–5192. - PMC - PubMed
    1. Mignogna, G., Simmaco, M., Kreil, G. & Barra, D. (1993) EMBO J. 12, 4829–4832. - PMC - PubMed
    1. Kamatani, Y., Minakata, H., Iwashita, T., Nomoto, K., In, Y., Doi, M. & Ishita, T. (1990) FEBS Lett. 276, 95–97. - PubMed

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