The multiple carrier model of nonribosomal peptide biosynthesis at modular multienzymatic templates.
Gramicidin S synthetase 1 and 2 were affinity-labeled at their thiolation centers either by thioesterification with the amino acid substrate or by specific alkylation with the thiol reagent N-ethylmaleimide in combination with a substrate protection technique. The labeled proteins were digested either chemically by cyanogen bromide or by proteases. An efficient multistep high pressure liquid chromatography methodology was developed and used to isolate the active site peptide fragments of all five thiolation centers of gramicidin S synthetase in pure form. The structures of these fragments are investigated by N-terminal sequencing, mass spectrometry, and amino acid analysis. Each of the active site peptide fragments contains the consensus motif LGG(H/D)S(L/I), which is specific for thioester formation in nonribosomal peptide biosynthesis. It was demonstrated that a 4'-phosphopantetheine cofactor is attached to the central serine of the thiolation motif in each amino acid-activating module of the gramicidin S synthetase multienzyme system forming the thioester binding sites for the amino acid substrates and catalyzing the elongation process. Our data are strong support for a "multiple carrier model" of nonribosomal peptide biosynthesis at multifunctional templates, which is discussed in detail.
Stein
T
T
Institut für Biochemie und Molekulare Biologie, Technische Universität Berlin, Franklinstrasse 29, D-10587 Berlin-Charlottenburg, Germany.
Vater
J
J
Kruft
V
V
Otto
A
A
Wittmann-Liebold
B
B
Franke
P
P
Panico
M
M
McDowell
R
R
Morris
H R
HR
D000591:Amino Acid Isomerases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001665:Binding Sites; D006096:Gramicidin / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D010453:Peptide Synthases / Q000378:metabolism*
D000596:Amino Acids; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D006096:Gramicidin; D000591:Amino Acid Isomerases; C022343:phenylalanine racemase (ATP-hydrolyzing); D010453:Peptide Synthases; C020831:gramicidin-S-synthetase 2