Detection of 4'-phosphopantetheine at the thioester binding site for L-valine of gramicidinS synthetase 2.
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.
Stein
T
T
Institut für Biochemie und Molekulare Biologie, Technische Universität Berlin, Germany.
Vater
J
J
Kruft
V
V
Wittmann-Liebold
B
B
Franke
P
P
Panico
M
M
Mc Dowell
R
R
Morris
H R
HR
D000591:Amino Acid Isomerases / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001407:Bacillus; D001665:Binding Sites; D004952:Esters; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D010204:Pantetheine / Q000031:analogs & derivatives* / Q000032:analysis; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism; D016339:Spectrometry, Mass, Fast Atom Bombardment; D014633:Valine / Q000737:chemistry* / Q000378:metabolism
D000596:Amino Acids; D004952:Esters; D009097:Multienzyme Complexes; D010204:Pantetheine; D000591:Amino Acid Isomerases; D010453:Peptide Synthases; C020831:gramicidin-S-synthetase 2; D014633:Valine; C003129:4'-phosphopantetheine