| Popis: |
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. The optimal reaction temperature for Trp chlorination is 45°C, and the melting temperatures of BorH and BorF are 48°C and 50°C respectively, which are higher than the thermal parameters for most other halogenases previously studied. BorH exhibits a broad substrate scope, chlorinating and brominating a variety of aromatic substrates with and without indole groups. 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. Quantitative conversion of L-tryptophan (Trp) to 6-chlorotryptophan could be achieved using 1.2 mol% BorH and 2 mol% BorF. 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. Four X-ray crystal structures of BorH alone and BorH complexed with Trp (substrate), 6-Cl-Trp (product), and FAD with Trp (cofactor and substrate) were solved. The BorH/Trp crystal structure shows the orientation of Trp positions C6 close to the catalytic Lys79 ε-amino group, providing a structural explanation for the observed regioselectivity. BorH’s thermal stability and its ability to chlorinate and brominate a variety of substrates make it a promising candidate for biocatalytic preparation of aryl halides. |
