BPS2026 – Teaching a nicotine-eating enzyme to breathe: A computational investigation into the evolution of oxidase activity in flavoenzymes

Nicotine oxidoreductase (NicA2) is a promising therapeutic enzyme for smoking cessation, but its clinical potential is hindered by poor catalytic efficiency. As a flavin-dependent dehydrogenase, NicA2 has an exceptionally slow turnover rate with molecular oxygen, requiring impractically large therapeutic doses. To overcome this, highly efficient variants have been developed through directed evolution (NicA2 v321) and ancestral sequence reconstruction, which resurrected a potent ancestral oxidase (Node D) from a slow dehydrogenase ancestor (Node C). While experimental methods provide crucial kinetic data, they cannot fully capture the dynamics of oxygen diffusion and mutational effect at an atomic resolution. This research employs molecular dynamics (MD) simulations as a “computational microscope” to provide time-resolved, mechanistic insights into how these enzymes evolved to “breathe.” We utilized enhanced sampled molecular dynamics (MD) simulations to investigate how specific mutations alter the conformational landscape and oxygen accessibility in NicA2 variants and ancestors. In the ancestral oxidase, mutations distal to the active site reduce global protein flexibility and volume, creating a more compact structure that optimally repositions a conserved lysine residue to stabilize the oxidative half-reaction. Conversely, in the directed evolution variant NicA2 v321, mutations potentially alter oxygen diffusion pathways, changing the accessibility and chemistry of the flavin-oxygen reaction. These mutations also facilitate oxygen diffusion to the flavin cofactor through an alternate pathway, only when the reaction product is bound. This computational analysis provides otherwise inaccessible, time-resolved detail, revealing how subtle amino-acid substitutions fine-tune flavin dynamics to control catalysis. These mechanistic insights are crucial for guiding the future rational design and optimization of NicA2 and other flavoenzymes for therapeutic and biotechnological applications.