Detection of 4'-phosphopantetheine at the thioester binding site for L-valine of gramicidinS synthetase 2

FEBS Lett. 1994 Feb 28;340(1-2):39-44. doi: 10.1016/0014-5793(94)80169-x.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Isomerases / chemistry*
  • Amino Acid Isomerases / metabolism
  • Amino Acid Sequence
  • Amino Acids / analysis
  • Bacillus
  • Binding Sites
  • Esters
  • Molecular Sequence Data
  • Multienzyme Complexes / chemistry*
  • Multienzyme Complexes / metabolism
  • Pantetheine / analogs & derivatives*
  • Pantetheine / analysis
  • Peptide Synthases / chemistry*
  • Peptide Synthases / metabolism
  • Spectrometry, Mass, Fast Atom Bombardment
  • Valine / chemistry*
  • Valine / metabolism

Substances

  • Amino Acids
  • Esters
  • Multienzyme Complexes
  • Pantetheine
  • Amino Acid Isomerases
  • Peptide Synthases
  • gramicidin-S-synthetase 2
  • Valine
  • 4'-phosphopantetheine