Characterization and Improvement of a Nicotine-Degrading Flavoenzyme

Abstract

Nicotine oxidoreductase (NicA2) is a flavin-dependent enzyme that has the ability to catalyze oxidation of nicotine to N-methylmyosmine. Though nicotine is a psychoactive substance with high addictive potential, N-methylmyosmine is not. Due to its ability to catalyze the breakdown of nicotine, NicA2 has been studied as a therapeutic to aid in the cessation of smoking. When NicA2 is injected intravenously or into the peritoneum of rats it reduces brain and bloodstream levels of nicotine, eases withdrawal symptoms, and cuts compulsive nicotine intake. The results of these preclinical studies are promising, but there is a major barrier to the use of this enzyme as a therapeutic in humans— the very high dose of enzyme that is required to achieve a positive response. This is likely due to the poor catalytic activity of NicA2 when only oxygen is supplied as an electron acceptor, as it can only degrade nicotine slowly under ambient conditions. My research uncovered that the reason for NicA2’s poor activity is a slow step in its reactive cycle, oxidation of flavin by dioxygen (O2). In the organism from which the nica2 gene was isolated, Pseudomonas putida S16, I discovered an alternative electron acceptor: a cytochrome c now named CycN. We have characterized the kinetic mechanism of the interaction between NicA2 and CycN and find that CycN oxidizes NicA2’s flavin at a rate ~10,000 times faster than O2 does. Additionally, I found that the cycN gene is required for the growth of P. putida S16 using nicotine as its carbon source. These data strongly support that CycN is the in vivo electron acceptor for NicA2. Though this cytochrome c allows NicA2 to achieve rapid turnover, is unlikely to be useful as an aid to smoking cessation. In order to make NicA2 a more effective therapeutic, the most direct approach is instead to increase its rate of reaction with O2, an oxidative substrate that is readily available in the bloodstream. This approach, however, is challenging: the molecular features that enable oxygen reactivity in flavoenzymes are enigmatic, and it has proven to be difficult to evolve NicA2 to be O2 reactive using rational substitutions based on our current understanding of flavoenzyme oxidation. I used directed evolution as an alternative approach, in part because it does not rely on a detailed mechanistic understanding of O2 activation. I randomly introduced mutations along the nicA2 gene and selected for variants that enable Pseudomonas putida S16 to grow aerobically on nicotine in the absence of CycN. This enabled me to isolate variant NicA2 proteins that have up to a 200-fold improvement in their ability to use oxygen as an electron acceptor. The mutations that enable this activity cluster within a solvent accessible tunnel that reaches into the active site of NicA2 and appear to modulate the accessibility of O2 to flavin. These improved variants are likely to be more effective as an aid to smoking cessation than wild type NicA2, as they should require a much lower dose to achieve the desired goal of nicotine degradation in the bloodstream.