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Updated: May 23, 2025

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Structurally guided engineering of flavin-dependent nicotine dehydrogenase.

Yuvarun Kapaothong1, Panu Pimviriyakul1

  • 1Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok, 10900, Thailand.

Archives of Biochemistry and Biophysics
|May 21, 2025
PubMed
Summary

Researchers engineered a nicotine-degrading enzyme (Nox-WT) to improve its efficiency for biodegradation and biodetection. A triple mutant (Nox-Y338F/H364V/W423H) demonstrated significantly enhanced nicotine transformation rates without compromising stability.

Keywords:
Enzyme engineeringFlavoenzymeModeled structureNicotineNicotine dehydrogenase

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Area of Science:

  • Biochemistry
  • Enzyme Engineering
  • Environmental Biotechnology

Background:

  • Nicotine, a toxic alkaloid from tobacco, pollutes the environment.
  • Existing nicotine biodegradation enzymes face limitations in efficiency and catalytic turnover.

Purpose of the Study:

  • To engineer a more efficient nicotine oxidase/dehydrogenase (Nox) for improved biodegradation and biodetection.
  • To overcome limitations such as slow oxygen reactions, incomplete flavin recovery, and substrate inhibition in wild-type Nox.

Main Methods:

  • Overexpression and purification of wild-type nicotine oxidase/dehydrogenase (Nox-WT) from Pseudomonas sp. HZN6.
  • Structural modeling using AlphaFold to identify sites for mutagenesis.
  • Site-directed mutagenesis and rapid kinetic techniques to screen and characterize enzyme variants.

Main Results:

  • Identified slow oxygen half-reaction as the rate-limiting step in Nox-WT.
  • Engineered a triple mutant (Nox-Y338F/H364V/W423H) with a 21-fold increase in FAD oxidation rate.
  • Eliminated substrate inhibition and achieved full flavin recovery in the mutant, enhancing overall catalytic efficiency.

Conclusions:

  • Rational enzyme engineering successfully improved nicotine transformation rates and efficiency.
  • The engineered Nox mutant offers enhanced performance for environmental applications like nicotine biodegradation and biodetection.
  • This study demonstrates the potential of enzyme engineering for developing sustainable solutions to environmental contamination.