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J Biol Chem, Vol. 273, Issue 29, 18011-18014, July 17, 1998

COMMUNICATION
Characterization of the Binding Site of the Tripeptide Intermediate D-PhenylalanylL-Prolyl-L-Valine in Gramicidin S Biosynthesis

Frank Leenders, Joachim Vater, Torsten Stein, and Peter Franke

From the  Max-Volmer-Institut für Biophysikalische Chemie und Biochemie, Technische Universität Berlin, Franklinstrasse 29, D-10587 Berlin, Germany and the  Institut für Biochemie, Freie Universität Berlin, Thielallee 63, D-14195 Berlin, Germany

The tripeptide intermediate D-Phe-Pro-Val in the biosynthesis of gramicidin S was labeled by incorporation of either L-[¹⁴C]phenylalanine or L-[¹⁴C]valine in an in vitro biosynthetic assay. The gramicidin S synthetase 2-tripeptide complex was first digested with CNBr and subsequently by Staphylococcus aureus V8 protease. The active site peptide carrying the radioactively labeled tripeptide was isolated in pure form by reversed phase high performance liquid chromatography technology and analyzed by liquid phase sequencing, mass spectrometry, and amino acid analysis. It was demonstrated that D-Phe-Pro-Val is attached to the 4'-phosphopantetheine cofactor at the thiolation center for valine of gramicidin S synthetase 2. In this way the attachment site of a peptide intermediate in nonribosomal peptide biosynthesis was identified for the first time. Our results are in full agreement with the multiple carrier model of nonribosomal peptide biosynthesis (Stein, T., Vater, J., Kruft, V., Otto, A., Wittmann-Liebold, B., Franke, P., Panico, M., McDowell, R., and Morris, H. R. (1996) J. Biol. Chem. 271, 15426-15435), which predicts that the growing peptide chain in the elongation process should always be bound to the thiotemplate site specific for its C-terminal amino acid component.

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