Zhang, Z.; Bandivadekar, P.; Gaunt, A.; Ahn, S.-H.; Barkman, T.; Stull, F.

Reduced flavin cofactors have the innate ability to reduce molecular oxygen to hydrogen peroxide. Flavoprotein oxidases turbocharge the reaction of their flavin cofactor with oxygen whereas flavoprotein dehydrogenases generally suppress it, yet our understanding of how these two enzyme classes control this reactivity remains incomplete. Here we used ancestral sequence reconstruction and biochemical characterization to retrace the evolution of oxidase activity in a lineage of nicotine/6-hydroxynicotine degrading enzymes of the flavoprotein amine oxidase superfamily. Our data suggest that the most ancient ancestor that gave rise to this lineage was a dehydrogenase, and that oxidase activity emerged later from within this group of dehydrogenases. We have identified the key amino acid replacements responsible for this emergence of oxidase activity, which, remarkably, span the entire protein structure. Molecular dynamics simulations indicate that this constellation of substitutions decreases the global dynamics of the protein in the evolution of oxidase function. This coincides with a dramatic restriction in the movement of a lysine residue in the active site, which more optimally positions it in front of the flavin to promote the reaction with O2. Our results demonstrate that sites distant from the flavin microenvironment can help control flavin-oxygen reactivity in flavoenzymes by modulating the conformational space and dynamics of the protein and catalytic residues in the active site.