Crystallographic and thermodynamic evidence of negative cooperativity of flavin and tryptophan binding in the flavin-dependent halogenases AbeH and BorH.

The flavin-dependent halogenase AbeH produces 5-chlorotryptophan in the biosynthetic pathway of the chlorinated bisindole alkaloid BE-54017. We report that , AbeH (assisted by the flavin reductase AbeF) can chlorinate and brominate tryptophan as well as other indole derivatives and substrates with phenyl and quinoline groups. We solved the X-ray crystal structures of AbeH alone and complexed with FAD, as well as crystal structures of the tryptophan-6-halogenase BorH alone, in complex with 6-chlorotryptophan, and in complex with FAD and tryptophan.

Steady-state kinetic analysis of halogenase-supporting flavin reductases BorF and AbeF reveals different kinetic mechanisms.

The short-chain flavin reductases BorF and AbeF reduce FAD to FADH, which is then used by flavin-dependent halogenases (BorH and AbeH respectively) to regioselectively chlorinate tryptophan in the biosynthesis of indolotryptoline natural products. Recombinant AbeF and BorF were overexpressed and purified as homodimers from E. coli, and copurified with substoichiometric amounts of FAD, which could be easily removed.