Mutational analysis of a type II thioesterase associated with nonribosomal peptide synthesis

Uwe Linne, Dirk Schwarzer, Gunnar N. Schroeder, Mohamed A. Marahiel*

*Corresponding author for this work

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

Recent studies on type II thioesterases (TEIIs) involved in microbial secondary metabolism described a role for these enzymes in the removal of short acyl-S-phosphopantetheine intermediates from misprimed holo-(acyl carrier proteins) and holo-(peptidyl carrier proteins) of polyketide synthases and nonribosomal peptide synthetases. Because of the absence of structural information on this class of enzymes, we performed a mutational analysis on a prototype TEII essential for efficient production of the lipopeptide antibiotic surfactin (TEIIsrf), which led to identification of catalytic and structural residues. On the basis of sequence alignment of 16 TEIIs, 10 single and one double mutant of highly conserved residues of TEIIsrf were constructed and biochemically investigated. We clearly identified a catalytic triad consisting of Ser86, Asp190 and His216, suggesting that TEIIsrf belongs to the αt/β-hydrolase superfamily. Exchange of these residues with residues with aliphatic side chains abolished enzyme activity, whereas replacement of the active-site Ser86 with cysteine produced an enzyme with marginally reduced activity. In contrast, exchange of the second strictly conserved asparagine (Asp163) with Ala resulted in an active but unstable enzyme, excluding a role for this residue in catalysis and suggesting a structural function. The results define three catalytic and at least one structural residue in a nonribosomal peptide synthetase TEII.

Original languageEnglish
Pages (from-to)1536-1545
Number of pages10
JournalEuropean Journal of Biochemistry
Volume271
Issue number8
DOIs
Publication statusPublished - Apr 2004
Externally publishedYes

Keywords

  • Catalytic triad
  • Fatty acid synthases
  • Nonribosomal peptide synthesis
  • Peptide synthetases
  • Type II thioesterase polyketide synthases

ASJC Scopus subject areas

  • Biochemistry

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