Beyond the Protein Matrix: Probing Cofactor Variants in a Baeyer–Villiger Oxygenation Reaction

A general question in biochemistry is the interplay between the chemical properties of cofactors and the surrounding protein matrix. Here, the functions of NADP⁺ and FAD are explored by investigation of a representative monooxygenase reconstituted with chemically modified cofactor analogues. Like pieces of a jigsaw puzzle, the enzyme active site juxtaposes the flavin and nicotinamide rings, harnessing their H-bonding and steric properties to finely construct an oxygen-reacting center that restrains the flavin peroxide intermediate in a catalytically competent orientation. Strikingly, the regio- and stereoselectivities of the reaction are essentially unaffected by cofactor modifications. These observations indicate a remarkable robustness of this complex multicofactor active site, which has implications for enzyme design based on cofactor engineering approaches