Detection of 4'‐phosphopantetheine at the thioester binding site for l‐valine of gramicidinS synthetase 2

FEBS Letters

Morris, Howard R.²  Stein, Torsten¹  Kruft, Volker³  Vater, Joachim¹  Dowell, Roy Mc²  Wittmann-Liebold, Brigitte³  Panico, Maria²  Franke, Peter⁴ 

[1] Institut für Biochemie und Molekulare Biologie, Technische Universität Berlin, Franklinstr. 29, D-10587 Berlin, Germany;Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AY, UK;Max-Delbrück-Zentrum für Molekulare Medizin, Robert-Rössle Str. 10. D-13122 Berlin-Buch, Germany;Institut für Biochemie, Freie Universität Berlin, Thielallee 63, D-14195 Berlin, Germany

关键词: GramicidinS synthetase;    Thioester binding site;    Affinity labeling;    Active site peptide;    4'-phosphopantetheine;    Multiple carrier model;    GS;    gramicidinS synthetase;    GS1;    gramicidinS synthetase 1 (phenylalanine racemase;    EC5.1.1.11);    GS2;    gramicidinS synthetase 2 (condensing;    peptide forming multienzyme);    Pan;    4'-phosphopantetheine;    NEM;    N-ethylmaleimide;    NES;    N-ethylsuccinimido;    x;    S-(N-ethylsuccinimido)4'-phosphopantetheine;    DTE;    dithioerythritol;    OPA;    o-phthaldialdehyde;    HPLC;    high-performance liquid chromatography;    FPLC;    fast protein (peptide) chromatography;    HF-FAB MS;    high-field fast-atom bombardment mass spectrometry;    ESI MS;    electrospray ionization mass spectrometry;    m/z;    mass to charge ratio;    kV;    kilovolt;   

DOI  :  10.1016/0014-5793(94)80169-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown