Directed evolution unlocks oxygen reactivity for a nicotine-degrading flavoenzyme

Abstract

The flavoenzyme nicotine oxidoreductase (NicA2) is a promising injectable treatment to aid in the cessation of smoking, a behavior responsible for one in ten deaths worldwide. NicA2 acts by degrading nicotine in the bloodstream before it reaches the brain. Clinical use of NicA2 is limited by its poor catalytic activity in the absence of its natural electron acceptor CycN. Without CycN, NicA2 is instead oxidized slowly by dioxygen (O2), necessitating unfeasibly large doses in a therapeutic setting. Here, we report a genetic selection strategy that directly links CycN-independent activity of NicA2 to growth of Pseudomonas putida S16. This selection enabled us to evolve NicA2 variants with substantial improvement in their rate of oxidation by O2. The encoded mutations cluster around a putative O2 tunnel, increasing flexibility and accessibility to O2 in this region. These mutations further confer desirable clinical properties. A variant form of NicA2 is tenfold more effective than the wild type at degrading nicotine in the bloodstream of rats.

A Robust Growth-Based Selection Platform to Evolve an Enzyme via Dependency on Noncanonical Tyrosine Analogues

“…Moreover, growth-based selections are cheap and straightforward to implement in the laboratory, as identifying improved biocatalysts typically involves either plating populations on selective media or serially (or continuously) culturing populations under selective conditions. In the latter scenario, more active biocatalysts will gradually outcompete less proficient ones (Figure D). − Lastly, previously employed selections for biocatalysts that directly confer a growth advantage to a host organism (e.g., chorismate mutase or β-lactamase) could be employed to identify enzyme variants with a wide range of activities. − …”