Abstract
Flavin-dependent halogenases (FDHs) have tremendous applications in synthetic chemistry. A single-component FDH, AetF, exhibits both halogenase and reductase activities in a continuous polypeptide chain. AetF exhibits broad substrate promiscuity and catalyzes the two-step bromination of l-tryptophan (l-Trp) to produce 5-bromotryptophan (5-Br-Trp) and 5,7-dibromo-l-tryptophan (5,7-di-Br-Trp). To elucidate the mechanism of action of AetF, we solved its crystal structure in complex with FAD, FAD/NADP+, FAD/l-Trp, and FAD/5-Br-Trp at resolutions of 1.92–2.23 Å. The obtained crystal structures depict the unprecedented topology of single-component FDH. Structural analysis revealed that the substrate flexibility and dibromination capability of AetF could be attributed to its spacious substrate-binding pocket. In addition, highly-regulated interaction networks between the substrate-recognizing residues and 5-Br-Trp are crucial for the dibromination activity of AetF. Several Ala variants underwent monobromination with >98 % C5-regioselectivity toward l-Trp. These results reveal the catalytic mechanism of single-component FDH for the first time and contribute to efficient FDH protein engineering for biocatalytic halogenation.
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