Catalytic roles of two serine residues in flavin-dependent quinone reductase HadB.

HadB is flavin mononucleotide (FMN)-dependent quinone reductase that plays a key role in preventing quinone accumulation during the biodegradation of halogenated phenols and nitrophenols. Although the reaction mechanism of HadB has been studied, its structure-function relationship remains unclear. In this study, the predicted structure of FMN-bound HadB, generated using AlphaFold version 3.0, revealed that it belongs to the oxygen (O2)-insensitive FMN-dependent nitroreductase family. FMN is bound at the dimer interface and is stabilized through intersubunit interactions. Site-directed mutagenesis was used to identify functionally important amino acid residues. Ser147, located near the N5 atom of FMN, is critical for FMN binding and may serve as the catalytic residue responsible for FMN reduction by NADH. The roles of adjacent residue, Ser43, from the neighboring subunit were also examined. The mutations of Ser43 to residues Ala and Val (Ser43Ala and Ser43Val) led to reduced quinone reductase activity, primarily due to a decrease in the FMN reduction rate by NADH during the reductive half-reaction. These structural insights into HadB's catalytic mechanism enhance our understanding of its function in the biodegradation pathway.