Structurally guided engineering of flavin-dependent nicotine dehydrogenase

Nicotine is a toxic alkaloid found in tobacco leaves that contaminates the environment when the leaves are smoked. In the present study, developed an enzyme technology for nicotine biodegradation and addressed biodetection applications. Recombinant wild-type nicotine oxidase/dehydrogenase from Pseudomonas sp. HZN6 (Nox-WT) was overexpressed and purified to ensure homogeneity. Nox-WT was clearly classified as a flavin adenine dinucleotide (FAD)-containing dehydrogenase, which catalyzes rapid nicotine oxidation in its reductive half-reaction; however, its oxidative half-reaction with O₂ was slow and was identified as the rate-limiting step. An imbalance in the rate between the two half-reactions limits the overall catalytic turnover of Nox-WT. According to kinetic behavior, incomplete flavin recovery and substrate inhibition were also identified as obstructed issues that limit the enzyme efficiency. Nox-WT engineering has been performed to address these problems. The modeled structure of Nox-WT was constructed using AlphaFold to design candidate residues for site-directed mutagenesis. Using systematic screening through rapid kinetic techniques, all the limitations were eliminated in the engineered triple mutated Nox-Y338F/H364V/W423H. The mutations at Y338F and H364V expanded the tunnel for O₂ accessibility, resulting in 21-fold faster FAD oxidation by O₂ in this mutant than in Nox-WT. Mutation at W423H disrupted the binding of nicotine; therefore, substrate inhibition was removed, and FAD was fully recovered. Nox-Y338F/H364V/W423H potentially transforms nicotine considerably faster than Nox-WT without loss of enzyme thermostability. Overall, using a rational design, we successfully engineered an effective mutant of Nox that would be useful for future applications.