GONUTS has been updated to MW1.31 Most things seem to be working but be sure to report problems.

Have any questions? Please email us at ecoliwiki@gmail.com

PMID:15743914

From GONUTS
Jump to: navigation, search
Citation

Yeh, E, Garneau, S and Walsh, CT (2005) Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 102:3960-5

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 k(cat) 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.

Links

PubMed PMC554827 Online version:10.1073/pnas.0500755102

Keywords

Carbazoles/metabolism; Chromatography, High Pressure Liquid; Flavin-Adenine Dinucleotide/analogs & derivatives; Flavin-Adenine Dinucleotide/metabolism; Indoles/metabolism; Kinetics; Oxidoreductases/metabolism; Tryptophan/analogs & derivatives; Tryptophan/biosynthesis; Tryptophan/chemistry

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

NOCAE:REBF

GO:0042602: flavin reductase activity

ECO:0000314:

F

Addition of RebF provides FADH2 for the halogenase to produce 7-chlorotryptophan during rebeccamycin biosynthesis


complete
CACAO 2542

NOCAE:REBF

enables

GO:0042602: riboflavin reductase (NADPH) activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete


See also

References

See Help:References for how to manage references in GONUTS.