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.

Structure and Activity of the Thermophilic Tryptophan-6 Halogenase BorH.

Flavin-dependent halogenases carry out regioselective aryl halide synthesis in aqueous solution at ambient temperature and neutral pH using benign halide salts, making them attractive catalysts for green chemistry. BorH and BorF, two proteins encoded by the biosynthetic gene cluster for the chlorinated bisindole alkaloid borregomycin A, are the halogenase and flavin reductase subunits of a tryptophan-6-halogenase. Quantitative conversion of l-tryptophan (Trp) to 6-chlorotryptophan could be achieved using 1.2 mol % BorH and 2 mol % BorF. The optimal reaction temperature for Trp chlorination is 45 °C, and the melting temperatures of BorH and BorF are 48 and 50 °C respectively, which are higher than the thermal parameters for most other halogenases previously studied. Steady-state kinetic analysis of Trp chlorination by BorH determined parameters of kcat =4.42 min-1 , and KM of 9.78 µm at 45 °C. BorH exhibits a broad substrate scope, chlorinating and brominating a variety of aromatic substrates with and without indole groups. Chlorination of Trp at a 100 mg scale with 52 % crude yield, using 0.2 mol % BorH indicates that industrial scale biotransformations using BorH/BorF are feasible. The X-ray crystal structure of BorH with bound Trp provides additional evidence for the model that regioselectivity is determined by substrate positioning in the active site, showing C6 of Trp juxtaposed with the catalytic Lys79 in the same binding pose previously observed in the structure of Thal.