Detection of 4'‐phosphopantetheine at the thioester binding site for l‐valine of gramicidinS synthetase 2

Abstract

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 [³H]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.