Bellizzi, J. J. | Search bioRxiv

bioRxiv2025

The X-ray crystal structure of BorF, the flavin reductase subunit of a two-component flavin-dependent tryptophan halogenase

BorF is a short-chain flavin reductase from a desert soil bacterium that uses NADH to reduce FAD to FADH2, which is used by the tryptophan-6-halogenase BorH to chlorinate tryptophan in the biosynthetic pathway of borregomycin A. The X-ray crystal structure of BorF bound to FAD was solved to 2.37 [A] by molecular replacement and consists of a homodimer of single-domain protomers with a Greek key split {beta}-barrel topology containing a domain-swapped N-terminal -helix, as seen in other members of this family. Insertions and deletions in the region between 3 and {beta}5 lead to a variety of different conformations of the adenosine portion of FAD bound to BorF and structurally related reductases. Comparison of the FAD-bound structures of BorF and BorH suggests that FAD must completely dissociate from BorH in order to be reduced by BorF.

biochemistry↗

bioRxiv2023

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 in vitro, 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. Partitioning of FAD and tryptophan into different chains of BorH and failure to incorporate tryptophan into AbeH/FAD crystals suggested that flavin and tryptophan binding are negatively coupled in both proteins. ITC and fluorescence quenching experiments confirmed the ability of both AbeH and BorH to form binary complexes with FAD or tryptophan and the inability of tryptophan to bind to AbeH/FAD or BorH/FAD complexes. FAD could not bind to BorH/tryptophan complexes, but FAD appears to displace tryptophan from AbeH/tryptophan complexes in an endothermic entropically-driven process.

biochemistry↗