Mechanistic basis for the low O2-dependent activity of a nicotine-degrading enzyme

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PROJECT SUMMARY/ABSTRACT Despite widespread knowledge of the negative health consequences of tobacco use, it is very challenging for tobacco users to cease using tobacco products due to the addictive properties of nicotine. A novel pharmacokinetic strategy that involves the administration of the nicotine-degrading enzyme, NicA2 from Pseudomonas putida S16, has recently been shown to be highly effective at reversing the symptoms of nicotine dependence in rodent models. NicA2 is a member of the flavin-containing amine oxidase family of enzymes, which invariably use O2 as an oxidant, and NicA2 is therefore also assumed to be an oxidase. However, the enzyme has displayed very poor O2-dependent catalytic activity, and this low activity has required the use of unacceptably high doses of NicA2 (10 mg/kg or greater) in order to achieve symptomatic relief of nicotine seeking behavior in animal models. We have discovered that O2 is not the natural oxidant for NicA2, explaining why the enzyme has displayed such low activity with O2; NicA2 is actually a dehydrogenase that uses a novel cytochrome c protein (CycN) as its immediate electron acceptor, making NicA2 a remarkable exception to the “oxidase” paradigm of the flavin-containing amine oxidase class of enzymes. This project is designed to elucidate the mechanistic underpinnings of NicA2’s use of CycN instead of O2. In the first aim, experimental evolution combined with genetic selections will be used to identify the structural features responsible for restricting NicA2’s capacity to use O2 as an electron acceptor. The second aim is designed to determine the mechanism of electron transfer between NicA2 and CycN. Site-directed mutagenesis and pre-steady state kinetics will be used to define the electron transfer pathway between the redox centers in the two proteins and site-specific chemical crosslinking will be used to determine the binding interface between NicA2 and CycN. The third aim will identify the downstream terminal electron acceptor(s) that reoxidize CycN in P. putida S16. The fundamental knowledge obtained through these efforts will provide a strong framework for the continued development of NicA2-based tobacco-cessation therapies, which could ultimately reduce the massive health burden associated with tobacco use. This project will also strengthen the research environment at a regional undergraduate institution that historically has not received substantial funding from the NIH and will expose undergraduate students to meritorious research involving an array of genetic, biochemical and microbiological techniques.