Interdomain Communication between the Thiolation and Thioesterase Domains of EntF Explored by Combinatorial Mutagenesis and Selection

Zhe Zhou, Jonathan R. Lai, Christopher T T. Walsh

Research output: Contribution to journalArticlepeer-review

30 Scopus citations

Abstract

Thiolation (T) domains are protein way stations in natural product assembly lines. In the enterobactin synthetase, the T domain on EntF is recognized in cis by its catalytic partners: the EntF condensation (C), adenylation (A), and thioesterase (TE) domains. To assess surface features of the EntF T domain recognized by C, A, and TE, regions of the EntF T domain were submitted to shotgun alanine scanning and Ent production selection, which revealed residues that could not be substituted by Ala. EntF mutants bearing Ala in such positions were assayed in vitro for Ent production with EntEB, and for A-T, C-T, and T-TE communications. We concluded that G1027A and M1030A are specifically defective in acyl transfer from T to TE. These residues define an interaction surface between these two in cis domains in an NRPS module.

Original languageEnglish (US)
Pages (from-to)869-879
Number of pages11
JournalChemistry and Biology
Volume13
Issue number8
DOIs
StatePublished - Aug 2006
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry

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