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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.