Mechanistic insights into nicotine oxidation by FAD-dependent nicotine dehydrogenase from Pseudomonas sp. HZN6.

Nicotine dehydrogenase from Pseudomonas sp. HZN6 (Nox) is a flavin-dependent enzyme that specifically catalyzes (S)-nicotine degradation through a relatively simple mechanism, making it a promising candidate for biotechnological applications. Although enzyme engineering has extensively targeted the oxidative half-reaction, key mechanistic details of the reductive half-reaction, particularly substrate oxidation, remain unclear. In this study, a three-dimensional model of the Nox-FAD-nicotine complex was constructed using AlphaFold in combination with molecular docking. This model enabled identification of putative catalytically relevant residues, specifically interaction network formed by residues E245, Y210, and Y214, located near the pyrrolidine nitrogen of (S)-nicotine. Site-directed mutagenesis combined with rapid kinetic analysis revealed that disruption of this network significantly decreased the rate of electron transfer from (S)-nicotine to FAD in the reductive half-reaction, as well as substrate binding affinity. Notably, these mutations did not affect the FAD oxidation with molecular oxygen during the oxidative half-reaction, which remains the rate-determining step. Consequently, the overall (S)-nicotine consumption activity was unaffected, even in variants where hydrogen-bonding capacity was completely abolished. These findings indicate that these residues are not strictly required as essential catalytic bases for (S)-nicotine oxidation. Instead, the active-site environment primarily facilitates proper substrate positioning and structural organization within the active site, thereby facilitating efficient catalysis.