Directed evolution unlocks oxygen reactivity for a nicotine-degrading flavoenzyme.

Area of Science:

• Biochemistry
• Enzyme Engineering
• Pharmacology

Background:

• Smoking cessation remains a global health challenge, with nicotine dependence being a primary driver.
• Nicotine oxidoreductase (NicA2) is an enzyme capable of degrading nicotine, showing therapeutic potential.
• Current limitations of NicA2 include poor activity without its natural electron acceptor, CycN, hindering clinical application.

Purpose of the Study:

• To engineer NicA2 variants with enhanced activity independent of CycN.
• To improve NicA2's catalytic efficiency using dioxygen (O2) as an alternative oxidant.
• To develop a more effective injectable treatment for smoking cessation.

Main Methods:

• A genetic selection strategy in *Pseudomonas putida* S16 was employed to identify NicA2 variants with improved CycN-independent activity.
• Directed evolution was used to enhance the enzyme's oxidation rate by O2.
• Structural analysis focused on mutations affecting a putative O2 tunnel.

Main Results:

• Evolved NicA2 variants demonstrated significantly improved oxidation rates by O2.
• Mutations identified clustered around a proposed O2 tunnel, increasing its flexibility and accessibility.
• A notable NicA2 variant exhibited tenfold greater efficacy in degrading bloodstream nicotine in rat models compared to the wild type.

Conclusions:

• The engineered NicA2 variants show enhanced catalytic activity and improved properties for therapeutic use.
• This work provides a pathway for developing more effective enzymatic treatments for nicotine addiction.
• The improved NicA2 variants represent a promising advancement in pharmacotherapy for smoking cessation.