Enzymology of surfactin biosynthesis

phosphopantetheinylation and aminoacylation of the peptide synthetase

L. E N Quadri, Peter Zuber, C. T. Walsh

Research output: Contribution to journalArticle

Abstract

Surfactin is a non-ribosomally synthesized cyclic lipoheptapeptide produced by Bacillus subtilis which has powerflfi biosurfaclant activity. Surfactin biosynthesis occurs by the so-called thiotemplate mechanism and is mediated by a peptide synthetase complex, surfactin synthetase. The inactive apo-surfactin synthetase is posttranslationally modified in each of seven amino acid activation domains with a 4'-phosphopantetheine prosthetic group to its active holo- form by Sfp, its cognate phosphopantetheinyl transferase. The reactions catalyzed by surfartiu synthetase, aminoacyladenytation, aminoacylenzyme thioester formation, and peptide bond formation, are shared by every other peptide synthetase and, together with lhe phosphopantetheinylation, are the key reactions in the thioternplate mechanism. We have overproduced in E. coli and purified the phosphopantetheinyl transferase Sfp and several domain fragments of surfactin synthetase. The in vitro stoichiometry, specificity, and kinetics of phosphopantetheinylation, aminoacyladenylation, and "in cis" and "in trans" aminoacylenzyme thioester formation by the fragments of the surfactin biosynthetic enzyme will be presented. Furthermore, we have performed site-directed mutagenesis on the sfp gene to probe the proposed phosphopantetheinyl transferase signature sequence. The kinetics of the phosphopantetheinyl transfer reaction and catalytic competence of the purified recombinant Sfp mutant proteins will be presented. The results validate the relevance of several residues in the transferase signature sequence.

Original languageEnglish (US)
JournalFASEB Journal
Volume11
Issue number9
StatePublished - 1997
Externally publishedYes

Fingerprint

surfactin
aminoacylation
Peptide Synthases
enzymology
Aminoacylation
Biosynthesis
ligases
biosynthesis
peptides
transferases
Mutagenesis
Kinetics
Bacilli
Mutant Proteins
Ligases
Transferases
Site-Directed Mutagenesis
Prosthetics
Bacillus subtilis
Stoichiometry

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Enzymology of surfactin biosynthesis : phosphopantetheinylation and aminoacylation of the peptide synthetase. / Quadri, L. E N; Zuber, Peter; Walsh, C. T.

In: FASEB Journal, Vol. 11, No. 9, 1997.

Research output: Contribution to journalArticle

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