Enabling Regiospecific Di-halogenation in One-Pot Reactions Using an Engineered Single-Component Flavin-Dependent Tryptophan Halogenase

The installation of halogen atoms into organic compounds
through
flavin-dependent halogenase (FDH)-catalyzed reactions has emerged
as an attractive strategy in organic biochemistry and synthetic biology.
A cyanobacterial FDH termed AetF that contains FDH and flavin reductase
in the same polypeptide chain could become a useful tool enzyme as
the need for including a reductase is omitted. Notably, AetF exploits
a specific substrate-interaction network to consecutively brominate
tryptophan (Trp) at C5 and then C7 to generate dibrominated Trp. In
this study, structure-based engineering is used to eliminate the C7-halogenation
capacity of AetF. The resulting variant, AetF-AIF, which contains
three residue substitutions, mainly catalyzes bromination or iodination
at C5 of Trp. We also show that combining AetF-AIF in tandem with
wild-type AetF enables the generation of heterogeneously halogenated
Trp in a one-pot reaction. The final products, 5-Br-7-I-Trp or 5-I-7-Br-Trp,
depending on the order of halide addition, account for a high ratio
in the reaction mixture without an additional purification step. These
results demonstrate the potential of single-component Trp-FDHs for
a wide range of halogenation reactions.