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

• Published in: Angewandte Chemie International Edition Vol. 63; no. 24; pp. e202403858 - n/a
• Main Authors: Prakinee, Kridsadakorn, Lawan, Narin, Phintha, Aisaraphon, Visitsatthawong, Surawit, Chitnumsub, Penchit, Jitkaroon, Watcharapa, Chaiyen, Pimchai
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 10.06.2024
• Edition: International ed. in English
• Subjects: De novo function; Electrophilic halogenation; Enzyme catalysis; Enzymes; Flavin; Flavin reductase; Flavin-dependent enzymes; Flavins - chemistry; Flavins - metabolism; Fluorimetry; Fluorometry; Halides; Halogenation; Hydrogen peroxide; Hydrogen Peroxide - chemistry; Hydrogen Peroxide - metabolism; Hydrolases - chemistry; Hydrolases - metabolism; Kinetics; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - metabolism; Mutagenesis; Organic compounds; Oxidoreductases - chemistry; Oxidoreductases - metabolism; Reaction mechanisms; Reductases; Spectrophotometry; Structural analysis; Substrates; Flavin-dependent enzymes; De novo function; Enzyme catalysis; Reaction mechanisms; Electrophilic halogenation
• ISSN: 1433-7851; 1521-3773; 1521-3773
• DOI: 10.1002/anie.202403858

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 (FlC4a‐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 FlC4a‐OOH in dehalogenases, selected hydroxylases and luciferases, but not in P2O can form HOX; however, the HOX generated from FlC4a‐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 FlC4a‐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. Electrophilic halogenases in nature are typically not efficient. Guided by flavin‐dependent halogenase mechanisms and taking advantage of the versatile reactivity of a flavin hydroperoxide adduct and in situ generation of H2O2 by flavin‐dependent enzymes, it was possible to promote the formation of a hypohalous acid—which is key for electrophilic halogenation—in various non‐native halogenases by rerouting the flavin‐generated peroxide.