Dichlorination of a pyrrolyl-S-carrier protein by FADH2- dependent halogenase PltA during pyoluteorin biosynthesis

Pieter C. Dorrestein, Ellen Yeh, Sylvie Garneau-Tsodikova, Neil L. Kelleher, Christopher T. Walsh*

*Corresponding author for this work

Research output: Contribution to journalArticle

128 Scopus citations

Abstract

The antifungal natural product pyoluteorin contains a 4,5-dichloropyrrole moiety. The timing of dichlorination in the heteroaromatic ring is now shown to occur after proline is tethered by thioester linkage to the carrier protein PltL and enzymatically desaturated to the pyrrolyl-S-PltL. Surprisingly, the FADH2-dependent halogenase PltA catalyzes chlorination at both positions of the ring, generating the 5-chloropyrrolyl-S-PltL intermediate and then the 4,5-dichloropyrrolyl-S-PltL product. PltA activity strictly depends on a heterologous flavin reductase that uses NAD(P)H to produce FADH2. Electrospray ionization-Fourier transform MS detected five covalent intermediates attached to the 11-kDa carrier protein PltL. Tandem MS localized the site of covalent modification on the carrier protein scaffold. HPLC analysis of the hydrolyzed products was consistent with the regiospecific chlorination at position 5 and then position 4 of the heteroaromatic ring. A mechanism for dichlorination is proposed involving formation of a FAD-4a-OCl intermediate for capture by the electron-rich C4 and C5 of the heteroaromatic pyrrole moiety.

Original languageEnglish (US)
Pages (from-to)13843-13848
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume102
Issue number39
DOIs
StatePublished - Sep 27 2005

Keywords

  • Clorobiocin
  • Coumermycin
  • Electrospray ionization-Fourier transform MS
  • Flavin reductase
  • Pyrrole biosynthesis

ASJC Scopus subject areas

  • General

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