A Critical Single-Point Mutation Enhancing the Catalytic Performance of Flavin-Dependent Halogenase RebH for Chlorination of Aminoquinolines

Abstract Flavin-dependent halogenases (FDHs) are promising biocatalysts capable of installing halogen atoms into aromatic and aliphatic compounds. However, the leakage of hypohalous acid (HOX), primarily caused by the imbalance between HOX generation and consumption rates, impairs their catalytic efficiency and limits practical applications. Herein, a structure-guided engineering strategy was performed to engineer FDH RebH for the efficient chlorination of aminoquinolines. A critical single-point variant, S110P, was screened via a facile high-throughput screening assay. Compared with the wild-type RebH, S110P exhibited 2.0- to 6.0-fold higher specific activity, 38%−94% enhanced catalytic efficiency, and significantly improved thermostability, with a 9.1 °C increase in melting temperature (Tm) and a 3.3-fold extended half-life (t1/2) at 35 °C. Moreover, S110P delivered 1.8- to 13.1-fold higher chlorination yields in 50 mL conversion reactions and showed a broadened substrate scope toward diverse quinolines. Computational analyses revealed that this single-point mutation modulated the conformational dynamics of key functional regions, strengthened HOCl binding in the active site, thereby reducing HOCl leakage by 73.5% over 30 min, and enhanced the propensity to form pre-reaction conformations. This work systematically elucidated the structure−function relationship of FDHs through mutational effect analysis of the critical site and laid an important foundation for their applications in biocatalytic halogenation of electron-deficient quinoline ring.