Fast Kinetics Reveals Rate-Limiting Oxidation and the Role of the Aromatic Cage in the Mechanism of the Nicotine-Degrading Enzyme NicA2

In Pseudomonas putida, the flavoprotein nicotine oxidoreductase (NicA2) catalyzes the oxidation of (S)-nicotine to N-methyl-myosmine, which is nonenzymatically hydrolyzed to pseudooxynicotine. Structural analysis reveals a monoamine oxidase (MAO)-like fold with a conserved FAD-binding domain and variable substrate-binding domain. The flavoenzyme has a unique variation of the classic aromatic cage with flanking residue pair W427/N462. Previous mechanistic studies using O-2 as the oxidizing substrate show that NicA2 has a low apparent K-m of 114 nM for (S)-nicotine with a very low apparent turnover number (k(cat) of 0.006 s(-1)). Herein, the mechanism of NicA2 was analyzed by transient kinetics. Single-site variants of W427 and N462 were used to probe the roles of these residues. Although several variants had moderately higher oxidase activity (7-12-fold), their reductive half-reactions using (S)-nicotine were generally significantly slower than that of wild-type NicA2. Notably, the reductive half-reaction of wild-type NicA2 is 5 orders of magnitude faster than the oxidative half-reaction with an apparent pseudo-first-order rate constant for the reaction of oxygen similar to k(cat). X-ray crystal structures of the N462V and N462Y/W427Y variants complexed with (S)-nicotine (at 2.7 and 2.3 angstrom resolution, respectively) revealed no significant active-site rearrangements. A second substrate-binding site was identified in N462Y/W427Y, consistent with observed substrate inhibition. Together, these findings elucidate the mechanism of a flavoenzyme that preferentially oxidizes tertiary amines with an efficient reductive half-reaction and a very slow oxidative half-reaction when O-2 is the oxidizing substrate, suggesting that the true oxidizing agent is unknown.

Automated summary

The research elucidated the mechanism of NicA2, showing its preference for oxidizing tertiary amines with an efficient reductive half-reaction and very slow oxidative half-reaction, suggesting that the true oxidizing agent is unknown.