Engineering Tryptophan Halogenase RebH for Regioselective Halogenation of Phenolic Compounds

Herein, we engineered tryptophan halogenase RebH using alanine scanning and the focused rational iterative site-specific mutagenesis (FRISM) strategy to expand its substrate scope toward phenolic compounds, particularly hydroxyquinolines and hydroxyisoquinolines, with excellent regioselectivity. The optimal mutant, 3-LSR/V80A/F111G, exhibited a 5-fold increase in catalytic activity and an 86.5-fold enhancement in catalytic efficiency (kcat/Km) for 6-hydroxyisoquinoline compared with the template 3-LSR. Furthermore, 3-LSR/V80A/F111G enabled the synthesis of 5-bromo-6-hydroxyisoquinoline on a milligram-scale with >99.9% conversion and 84.3% isolated yield.