TY - JOUR
T1 - The modular organization of multifunctional peptide synthetases
AU - Vater, Joachim
AU - Stein, Torsten
AU - Vollenbroich, Dirk
AU - Kruft, Volker
AU - Wittmann-Liebold, Brigitte
AU - Franke, Peter
AU - Liu, Li
AU - Zuber, Peter
PY - 1997
Y1 - 1997
N2 - 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.
AB - 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.
KW - Active site mutagenesis
KW - Electrospray mass spectrometry
KW - Modular structure
KW - Multiple 4'-phosphopantetheine cofactors
KW - Peptide synthetases
KW - Thioester binding site
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U2 - 10.1023/A:1026386100259
DO - 10.1023/A:1026386100259
M3 - Article
C2 - 9246644
AN - SCOPUS:53249145471
SN - 0277-8033
VL - 16
SP - 557
EP - 564
JO - Journal of Protein Chemistry
JF - Journal of Protein Chemistry
IS - 5
ER -