The modular organization of multifunctional peptide synthetases.
Gramicidin S synthetase 2 from B. brevis was affinity labeled at its valine thiolation center with the thiol reagent N-[3H]ethylmaleimide. From a tryptic digest of the enzyme-inhibitor complex a radioactive fragment was isolated in pure form by two reversed-phase HPLC steps. It was identified by liquid-phase N-terminal sequencing in combination with electrospray mass spectrometry (ESI-MS) as a hexadecapeptide containing the thiolation motif LGG(H/D)S(L/I). By ESI-MS it was demonstrated that a 4'-phosphopantetheine cofactor was attached to this fragment at its reactive serine. These results are consistent with the "Multiple Carrier Model" of nonribosomal peptide biosynthesis. Site-specific mutagenesis has been performed in thiolation, elongation, and epimerization motifs of some of the modules of surfactin synthetase from B. subtilis to clarify the function of prominent conserved amino acid residues in the intermediate steps of peptide biosynthesis. The modular structure of multifunctional peptide synthetases is discussed.
Vater
J
J
Max-Volmer-Institut für Biophysikalische Chemie und Biochemie, Technische Universität Berlin, Germany. jovajceh@mailszrz_zrz_TU-Berlin.DE
Stein
T
T
Vollenbroich
D
D
Kruft
V
V
Wittmann-Liebold
B
B
Franke
P
P
Liu
L
L
Zuber
P
P
D000591:Amino Acid Isomerases / Q000737:chemistry* / Q000502:physiology*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000502:physiology*; D010453:Peptide Synthases / Q000737:chemistry* / Q000502:physiology*; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D000591:Amino Acid Isomerases; D010453:Peptide Synthases; C020831:gramicidin-S-synthetase 2; C069542:surfactin synthetase