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Detection of 4'‐phosphopantetheine at the thioester binding site for l ‐valine of gramicidinS synthetase 2
Author(s) -
Stein Torsten,
Vater Joachim,
Kruft Volker,
Wittmann-Liebold Brigitte,
Franke Peter,
Panico Maria,
Dowell Roy Mc,
Morris Howard R.
Publication year - 1994
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(94)80169-x
Subject(s) - nonribosomal peptide , thioester , valine , biochemistry , biosynthesis , peptide , stereochemistry , serine , amino acid , active site , peptide sequence , acyl carrier protein , chemistry , binding site , biology , enzyme , gene
Biosynthesis of gramicidinS in Bacillus brevis is catalysed by a multienzyme system consisting of two multifunctional proteins, gramicidinS synthetase 1 and 2 codified by the grsA and grsB genes, respectively. GramicidinS synthetase 2 shows a modular architecture of four amino acid‐activating domains each containing a thioester binding motif LGG H/D S L/I highly conserved in its C‐terminal region, as demonstrated by sequence analysis of the grsB gene [W. Schlumbohm et al. (1991) J. Biol. Chem. 266, 23135‐23141]. This multienzyme was specifically labeled at the thioester binding site of l ‐valine with [ 3 H] N ‐ethylmaleimide using a substrate protection technique. After enzymatic digestion a labeled active site peptide was isolated in pure form by multistep methodology. This fragment was identified by gas‐phase sequencing as the active site peptide of the thiotemplate site for l ‐Val by comparison with the grsB gene sequence. By mass spectrometry in combination with amino acid analysis it was demonstrated that a 4'‐phosphopantetheine carrier was attached to the active serine in this motif. Our results give evidence that multiple peripheral 4'‐phosphopantetheine carriers are involved in the formation of gramicidinS in contrast to a central carrier arm as assumed in the original version of the thiotemplate mechanism. A ‘Multiple Carrier Model’ of nonribosomal peptide biosynthesis is proposed.