Fighting Smoking with Science: Evolving a Nicotine-Eating Enzyme

Smoking remains a massive global health crisis, causing one in ten deaths worldwide. While many people want to quit, the intense grip of nicotine addiction makes it incredibly difficult, with roughly 80% of attempts ending in relapse. In our latest study with the Bardwell lab at HHMI, we used the power of "directed evolution" to supercharge a bacterial enzyme that can eat nicotine in the blood before it ever reaches the brain.

The Big Question

We focused on an enzyme called NicA2, which naturally breaks down nicotine. The idea is simple: if we inject this enzyme into the bloodstream, it can intercept nicotine from a cigarette and neutralize it. However, there was a major hurdle. In its original form, NicA2 is very slow at using oxygen to complete its chemical reaction. Because of this "poor breathing," we previously had to use massive, unfeasible doses to see any benefit in animal trials. We wanted to know if we could "evolve" this enzyme in the lab to make it react with oxygen thousands of times faster.

The Study

We created a clever genetic selection system using a specific strain of bacteria (Pseudomonas putida). We engineered these bacteria so they could only grow if the NicA2 enzyme was working efficiently using oxygen. By creating millions of slightly different "mutant" versions of the enzyme and letting only the most active ones survive, we forced the enzyme to evolve better oxygen reactivity.

What We Discovered

Our laboratory evolution was a major success, yielding enzyme variants that were significantly more powerful than the original:

• Massive Speed Increase: We isolated variants with up to a 189 fold improvement in their catalytic rate.

  
  

• Unlocking the Oxygen Tunnel: We discovered that the best mutations clustered around a tiny "tunnel" in the enzyme's structure. These changes made the tunnel more flexible and accessible, allowing oxygen to reach the enzyme's core much more easily.

  
  

• Success in Living Subjects: When we tested a top variant (v321) in rats, it was ten times more effective at degrading nicotine than the wild type enzyme.

  
  

• Undetectable Nicotine: At its highest dose, the evolved enzyme maintained nicotine at undetectable levels in the blood, effectively creating a "chemical shield".

  
  

Why It Matters

This research is a vital step toward a new kind of smoking cessation therapy. By making the enzyme ten times more potent, we have brought the required dose down to a range that could actually work as a practical human injection.

The goal is to provide a tool that neutralizes the reward of smoking by clearing nicotine from the body within seconds. This could help break the cycle of addiction for people who have struggled with traditional quitting methods. While more testing is needed, we believe this evolved enzyme offers an exciting path toward helping millions of people achieve long term abstinence from tobacco.

Reference: Dulchavsky, M., Mitra, R., Wu, K., Li, J., Boer, K., Liu, X., Zhang, Z., Vasquez, C., Clark, C.T., Funckes, K., Shankar, K., Bonnet-Zahedi, S., Siddiq, M., Sepulveda, Y., Suhandynata, R.T., Momper, J.D., Calabrese, A.N., George, O., Stull, F., & Bardwell, J.C.A. (2023). Directed evolution unlocks oxygen reactivity for a nicotine-degrading flavoenzyme. Nature Chemical Biology, 19(11), 1406-1414. https://doi.org/10.1038/s41589-023-01426-y