Abstract
<jats:p>Engineering photoenzymes to perform new-to-nature reactions is a promising route towards green and sustainable photobiocatalysts. Fatty acid photodecarboxylases (FAPs) have been recently repurposed to catalyze radical cyclization reactions, but precise mechanistic information about how mutations redirect native activity is still lacking. Here, we combine QM/MM and ML/MM dynamic simulations to investigate the three key steps leading to cyclic products in a FAP mutant, previously engineered through directed-evolution techniques. We show that mutations enlarging the enzyme active site involve a delicate trade-off: they partially hinder the initial forward electron transfer, yet are simultaneously essential for enabling the subsequent cyclization step. Rather than statically pre-organizing the active site, the engineered enzyme adaptively confines the radical intermediate into distinct conformations at each step of catalysis, granting it access to productive cyclization pathways and achieving the experimentally observed ~90% cyclization yield. These findings establish adaptive confinement as a key determinant of new reactivity and provide a mechanistic framework for the rational engineering of next-generation photobiocatalysts.</jats:p>