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AssigneesThe Regents of the University of Michigan, The Board of Trustees of Western Michigan University
InventorsJames C. Bardwell, Mark Dulchavsky, Frederick Stull
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The disclosure provides variants of nicotine oxidoreductase and methods to select such variants that are unexpectedly active in the catabolic destruction of nicotine by oxidation using oxygen as electron acceptor, and catabolically active fragments thereof. Also disclosed are compositions comprising the CycN cytochrome c protein and at least one of the variant nicotine oxidoreductase holoenzymes, the fragments thereof, or a naturally occurring nicotine oxidoreductase, as well as fusion proteins comprising catalytically active nicotine oxidoreductase fragments or holoenzymes and CycN cytochrome c fragments or holoenzymes. Additionally, variants of L-6-hydroxynicotine oxidase, or catalytically active fragments thereof, are provided. Further disclosed are polynucleotides encoding such proteins, vectors comprising such polynucleotides, and host cells comprising such polynucleotides or vectors. Also provided are methods of using any of the disclosed compositions or formulations to treat nicotine dependence or reduce the risk of relapse to nicotine dependence.
A NicA2 nicotine oxidoreductase variant or functional fragment thereof comprising fewer than 10 amino acid substitutions, additions, or deletions from the amino acid sequence set forth in SEQ ID NO:131, wherein the NicA2 nicotine oxidoreductase variant exhibits a higher KM and/or a higher Kcat for oxidizing nicotine with oxygen as electron acceptor compared to the wild-type NicA2 nicotine oxidase of SEQ ID NO:131.
A method of identifying a nicotine oxidoreductase (NicA2) variant using O2 as an electron acceptor comprising:
A method of reducing nicotine dependence in a subject comprising administering a therapeutically effective amount of
A high throughput screen for a NicA2 nicotine oxidoreductase variant using O2 as electron acceptor comprising:
Dependencies are inferred from the claim text.