Characterization of the binding site of the tripeptide intermediate D-Phenylalanyl L-prolyl-L-valine in gramicidin S biosynthesis.
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.
Leenders
F
F
Max-Volmer-Institut für Biophysikalische Chemie und Biochemie, Technische Universität Berlin, Franklinstrasse 29, D-10587 Berlin, Germany.
Vater
J
J
Stein
T
T
Franke
P
P
D000591:Amino Acid Isomerases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001407:Bacillus; D001665:Binding Sites; D006096:Gramicidin / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010453:Peptide Synthases / Q000378:metabolism*; D010649:Phenylalanine; D011392:Proline; D012697:Serine Endopeptidases / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013211:Staphylococcus aureus; D014633:Valine
D009097:Multienzyme Complexes; D009842:Oligopeptides; D010446:Peptide Fragments; D006096:Gramicidin; D010649:Phenylalanine; D011392:Proline; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D000591:Amino Acid Isomerases; C022343:phenylalanine racemase (ATP-hydrolyzing); D010453:Peptide Synthases; C020831:gramicidin-S-synthetase 2; D014633:Valine