On the Mechanisms of Hypohalous Acid Formation and Electrophilic Halogenation by Non-Native Halogenases

Enzymatic electrophilic halogenation is a mild tool for functionalization of diverse organic compounds. Only a few groups of native halogenases are capable of catalyzing such a reaction. In this study, we used a mechanism-guided strategy to discover the electrophilic halogenation activity catalyzed by non-native halogenases. As the ability to form a hypohalous acid (HOX) is key for halogenation, flavin-dependent monooxygenases/oxidases capable of forming C4a-hydroperoxyflavin (Fl_C4a-OOH), such as dehalogenase, hydroxylases, luciferase and pyranose-2-oxidase (P2O), and flavin reductase capable of forming H₂O₂ were explored for their abilities to generate HOX in situ. Transient kinetic analyses using stopped-flow spectrophotometry/fluorometry and product analysis indicate that Fl_C4a-OOH in dehalogenases, selected hydroxylases and luciferases, but not in P2O can form HOX; however, the HOX generated from Fl_C4a-OOH cannot halogenate their substrates. Remarkably, in situ H₂O₂ generated by P2O can form HOI and also iodinate various compounds. Because not all enzymes capable of forming Fl_C4a-OOH can react with halides to form HOX, QM/MM calculations, site-directed mutagenesis and structural analysis were carried out to elucidate the mechanism underlying HOX formation and characterize the active site environment. Our findings shed light on identifying new halogenase scaffolds besides the currently known enzymes and have invoked a new mode of chemoenzymatic halogenation.