A protein interaction surface in nonribosomal peptide synthesis mapped by combinatorial mutagenesis and selection

Jonathan R. Lai, Michael A. Fischbach, David R. Liu, Christopher T. Walsh

Research output: Contribution to journalArticle

50 Citations (Scopus)

Abstract

Nonribosomal peptide synthetases (NRPSs) and polyketide synthases are large, multidomain enzymes that biosynthesize a number of pharmaceutically important natural products. The recognition of biosynthetic intermediates, displayed via covalent attachment to carrier proteins, by catalytic domains is critical for NRPS and polyketide synthase function. We report the use of combinatorial mutagenesis coupled with in vivo selection for the production of the Escherichia coli NRPS product enterobactin to map the surface of the aryl carrier protein (ArCP) domain of EntB that interacts with the downstream elongation module EntF. Two libraries spanning the predicted helix 2 and loop 2/helix 3 of EntB-ArCP were generated by shotgun alanine scanning and selected for their ability to support enterobactin production. From the surviving pools, we identified several hydrophobic residues (M249, F264, and A268) that were highly conserved. These residues cluster near the phosphopantetheinylated serine in a structural model, and two of these positions are in the predicted helix 3 region. Subsequent in vitro studies are consistent with the hypothesis that these residues form a surface on EntB required for interaction with EntF. These results suggest that helix 3 is a major recognition element in EntB-ArCP and demonstrate the utility of selection-based approaches for studying NRPS biosynthesis.

Original languageEnglish (US)
Pages (from-to)5314-5319
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume103
Issue number14
DOIs
StatePublished - Apr 4 2006
Externally publishedYes

Fingerprint

Peptide Synthases
Mutagenesis
Carrier Proteins
Membrane Proteins
Enterobactin
Polyketide Synthases
Peptides
Nucleic Acid-Independent Peptide Biosynthesis
Structural Models
Firearms
Biological Products
Alanine
Serine
Libraries
Catalytic Domain
Escherichia coli
Enzymes
Protein Domains
non-ribosomal peptide synthase

Keywords

  • Nonribosomal peptide synthetase
  • Siderophore

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

A protein interaction surface in nonribosomal peptide synthesis mapped by combinatorial mutagenesis and selection. / Lai, Jonathan R.; Fischbach, Michael A.; Liu, David R.; Walsh, Christopher T.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 103, No. 14, 04.04.2006, p. 5314-5319.

Research output: Contribution to journalArticle

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