RebH

RebH is a flavin-dependent tryptophan halogenase encoded by the RebH gene within the rebeccamycin biosynthetic gene cluster in certain soil bacteria. RebH catalyzes the regioselective chlorination of L-tryptophan at the 7-position, producing 7-chlorotryptophan. The enzyme works in conjunction with a separate flavin reductase, RebF, which supplies the reduced flavin (FADH2) required for halogenation.

In the catalytic cycle, RebF reduces FAD to FADH2; RebH uses FADH2 to convert chloride and oxygen into a reactive halogenating species that is delivered to the substrate in the active site, enabling electrophilic aromatic substitution on the indole ring.

RebH has a relatively narrow natural substrate scope, primarily acting on L-tryptophan, but it can accept certain tryptophan analogs and other indole derivatives. Through directed evolution and protein engineering, RebH variants have been developed with altered regiospecificity, broader substrate tolerance, improved activity, and compatibility with nonnative substrates, enabling 5- or 6-chlorination or bromination under certain conditions.

RebH belongs to the family of flavin-dependent halogenases, sharing structural features with other bacterial FDHs; crystal structures and mutational data have illuminated active-site residues that govern substrate binding and regioselectivity. The RebF reductase is encoded nearby in the same gene cluster and supplies FADH2.

RebH is used as a biocatalyst for the selective synthesis of 7-chloroindole derivatives, including 7-chlorotryptophan, serving as precursors in the biosynthesis and semi-synthesis of halogenated natural products and pharmaceutical leads. It is a model system for studying enzyme-based halogenation and for evolving new halogenation catalysts.