Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis

Ellen Yeh, Sylvie Garneau, Christopher T. Walsh

Research output: Contribution to journalArticlepeer-review

232 Scopus citations

Abstract

The indolocarbazole antitumor agent rebeccamycin is modified by chlorine atoms on each of two indole moieties of the aglycone scaffold. These halogens are incorporated during the initial step of its biosynthesis from conversion of L-Trp to 7-chlorotryptophan. Two genes in the biosynthetic cluster, rebF and rebH, are predicted to encode the flavin reductase and halogenase components of an FADH2-dependent halogenase, a class of enzymes involved in the biosynthesis of numerous halogenated natural products. Here, we report that, in the presence of O2, chloride ion, and L-Trp as cosubstrates, purified RebH displays robust regiospecific halogenating activity to generate 7-chlorotryptophan over at least 50 catalytic cycles. Halogenation by RebH required the addition of RebF, which catalyzes the NADH-dependent reduction of FAD to provide FADH2 for the halogenase. Maximal rates were achieved at a RebF/RebH ratio of 3:1. In air-saturated solutions, a kcat of 1.4 min-1 was observed for the RebF/RebH system but increased at least 10-fold in low-pO2 conditions. RebH was also able to use bromide ions to generate monobrominated Trp. The demonstration of robust chlorinating activity by RebF/RebH sets up this system for the probing of mechanistic questions regarding this intriguing class of enzymes.

Original languageEnglish
Pages (from-to)3960-3965
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume102
Issue number11
DOIs
StatePublished - Mar 15 2005

Keywords

  • Enzymology
  • Flavoenzyme
  • Natural product biosynthesis

ASJC Scopus subject areas

  • General

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