The flavoprotein L-hydroxynicotine oxidase (LHNO) catalyzes an early step in the bacterial catabolism of nicotine. Although, the structure of the enzyme establishes that it is a member of the monoamine oxidase family, LHNO is generally accepted to oxidize a carbon-carbon bond in the pyrrolidine ring of the substrate and has been proposed to catalyze the subsequent tautomerization and hydrolysis of the initial oxidation product to yield 6-hydroxypseudooxynicotine (Kachalova et al. (2011) Proc. Natl. Acad. Sci. USA 108, 4800–4805). Analysis of the product of the enzyme from Arthrobacter nicotinovorans by NMR and continuous-flow mass spectrometry establishes that the enzyme catalyzes the oxidation of the pyrrolidine carbon-nitrogen bond, the expected reaction for a monoamine oxidase, and that hydrolysis of the amine to form 6-hydroxypseudooxynicotine is nonenzymatic. Based on the kcat/Km and kred values for (S)-hydroxynicotine and several analogs, the methyl group contributes only marginally (~0.5 kcal/mol) to transition state stabilization, while the hydroxyl oxygen and pyridyl nitrogen each contribute ~4 kcal/mol. The small effects on activity of mutagenesis of His187, Glu300, or Tyr407 rule out catalytic roles for all three of these active-site residues.

The flavoenzyme L-6-hydroxynicotine oxidase (LHNO) is a member of the monoamine oxidase family that catalyzes the oxidation of (S)-6-hydroxynicotine to 6-hydroxypseudooxynicotine during microbial catabolism of nicotine. While the enzyme has long been understood to catalyze oxidation of carbon-carbon bond, it has recently been shown to catalyze oxidation of a carbon-nitrogen bond (Fitzpatrick et al., Biochemistry 55, 697–703). The effects of pH and mutagenesis of active site residues have now been utilized to study the mechanism and the roles of active site residues. Asn166 and Tyr311 bind the substrate, while Lys287 forms a water-mediated hydrogen bond with the flavin N(5). The N166A and Y311F mutations result in decreases of ~30 and ~4-fold in the kcat/Km and kred values for (S)-6-hydroxynicotine, respectively, with larger effects on the kcat/Km value for (S)-6-hydroxynornicotine. The K287M mutation results in a decrease of ~10-fold in these parameters and a 6,000-fold in the kcat/Km value for oxygen. The shapes of the pH profiles are not altered by the N166A and Y311F mutations. There is no solvent isotope effect on the kcat/Km value for amines. The results are consistent with a model in which both the charged and neutral forms of the amine can bind, with the former rapidly losing a proton to a hydrogen bond network of water and amino acids in the active site prior to hydride transfer to the flavin.