Flavin-dependent enzymes as multifunctional biocatalysts: A mechanistic perspective

Flavin-dependent enzymes constitute one of the most versatile classes of biocatalysts, owing to the rich redox chemistry and multifunctionality of flavin cofactors. Beyond their well-established roles in natural oxidation-reduction processes, recent advances have revealed that flavoproteins exhibit remarkable catalytic promiscuity, enabling a wide array of non-native transformations that extend well beyond their canonical reaction space. In this review, we present a comprehensive and mechanistically oriented overview of the catalytic promiscuity of flavin-dependent enzymes. We systematically categorize flavin enzyme reactivity into natural mechanism-based promiscuity, in which native catalytic cycles are repurposed for non-native reactions, and natural-mechanism-distinct promiscuity, where fundamentally new reaction pathways are unlocked through strategies such as cofactor engineering, protein engineering, reaction-condition modulation, additive effects, or photoactivation. Representative flavin-dependent enzyme families-including ene-reductases, flavoprotein monooxygenases, GMC-type oxidases, nitroreductases, monoamine oxidases and flavin-dependent halogenases-are discussed in detail, with emphasis on mechanistic principles governing substrate activation, redox-state control, and stereochemical outcomes. By highlighting unifying mechanistic concepts and emerging design strategies, this review aims to provide a conceptual framework to guide the future development of flavin-dependent biocatalysis toward increasingly complex, selective, and non-natural chemical transformations.