A programmable bifunctional flavoenzyme for direct amine-to-ester conversion

The unification of mechanistically distinct oxidative transformations within a single enzyme active site represents a long-standing challenge in biocatalysis. In particular, flavin-dependent oxidative deamination and Baeyer-Villiger oxidation have remained evolutionarily and mechanistically segregated, raising fundamental questions as to whether their catalytic cycles can be coherently integrated without mutual interference. Here, we report a bifunctional ancestral flavoenzyme, AncFO-221, reconstructed using a function-oriented ancestral sequence reconstruction strategy, which enables direct amine-to-ester conversion within a single active site. Combined experimental and computational analyses reveal a unified catalytic framework in which histidine-assisted proton and hydride transfer during amine oxidation is intrinsically coupled to C_4a-peroxyflavin-mediated oxygen insertion, establishing a continuous amine oxidation-Baeyer-Villiger oxidation (AO-BVO) reaction cycle rather than a fortuitous cascade. Guided by this mechanistic unity, modular protein engineering produced an optimized variant, M15, exhibiting an ~18-fold enhancement in catalytic efficiency, near-complete suppression of reductive side reactions, and lactone yields up to 93%. Notably, the engineered enzyme displays programmable and unconventional regioselectivity, preferentially migrating weakly migratory groups across structurally diverse amines, thereby overriding the classical Baeyer-Villiger migratory rule. This study demonstrates that ancestral reconstruction combined with mechanism-guided evolution can merge evolutionarily segregated chemistries into a single, tunable catalytic platform, providing a generalizable blueprint for the design of multistep oxidative biocatalysts.