Altering 2‐Hydroxybiphenyl 3‐Monooxygenase Regioselectivity by Protein Engineering for the Production of a New Antioxidant

2‐Hydroxybiphenyl 3‐monooxygenase is a flavin‐containing NADH‐dependent aromatic hydroxylase that oxidizes a broad range of 2‐substituted phenols. In order to modulate its activity and selectivity, several residues in the active site pocket were investigated by saturation mutagenesis. Variant M321A demonstrated altered regioselectivity by oxidizing 3‐hydroxybiphenyl for the first time, thus enabling the production of a new antioxidant, 3,4‐dihydroxybiphenyl, with similar ferric reducing capacity to the well‐studied piceatannol. The crystal structure of M321A was determined (2.78 Å), and molecular docking of the 3‐substituted phenol provided a rational explanation for the altered regioselectivity. Furthermore, HbpA was found to possess pro‐S enantioselectivity towards the production of several chiral sulfoxides, whereas variant M321F exhibited improved enantioselectivity. Based on the biochemical characterization of several mutants, it was suggested that Trp97 stabilized the substrate in the active site, Met223 was involved in NADH entrance or binding to the active site, and Pro320 might facilitate FAD movement.