Mechanism of the Flavoprotein l-Hydroxynicotine Oxidase: Kinetic Mechanism, Substrate Specificity, Reaction Product, and Roles of Active-Site Residues

Abstract

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.

Mechanism of Flavoprotein l-6-Hydroxynicotine Oxidase: pH and Solvent Isotope Effects and Identification of Key Active Site Residues

“…This suggests that both the protonated and the unprotonated forms of substrates can bind productively to LHNO. The only residue in the active site of LHNO with the potential to act as a base to accept a proton from the substrate is Tyr407; the lack of a significant decrease in activity for the Y407F enzyme 5 rules out this residue as the sole source of the pK a . In the structure of the complex of LHNO with (S)-6-hydroxynicotine (Figure S3), the pyrrolidine nitrogen is 3.2 Å from a water molecule 21 .…”

“…(S)-4-Hydroxynornicotine was from Asiba Pharmatech Inc. (Milltown, NJ). Site-directed mutagenesis of pETHLNO 5 was carried out using the QuikChange protocol (Stratagene). Wild-type and variant LHNOs from Arthrobacter nitotinovorans were expressed in Escherichia coli and purified as previously described 5 .…”

“… * Conditions: 0.1 M Hepes (pH 8), 0.1 M NaCl, at 25 ° C. a The values for the wild-type enzyme are from reference 5. b Determined by varying the concentration of the amine with 1.2 mM oxygen. c The kinetic parameters for (S)-6-hydroxynornicotine were determined by varying the concentration of the amine in air-saturated buffer, so that the k cat and K m values should be considered apparent values. …”

“…There is a great deal of structural information regarding LHNO, including structures of the enzyme with substrates and products 2, 3 , but there has been little study of the mechanism. While the enzyme was long assumed to catalyze the oxidation of a carbon-carbon bond in its substrate 4 , we recently established that LHNO catalyzes oxidation of a carbon-nitrogen bond (Scheme 1) 5 . Amine oxidation by LHNO is consistent with the protein structure, in that it is a member of the monoamine oxidase (MAO) structural family of flavoproteins 3 .…”

Structural Analysis Provides Mechanistic Insight into Nicotine Oxidoreductase from Pseudomonas putida

“…Evidence has been provided for the mechanism of oxidation of (S)-6-OH-nicotine catalyzed by 6HLNO as a two-step reaction: the flavin-dependent formation of 6-OH-N-methyl-myosmine followed by hydrolysis to form 6-OH-pseudooxynicotine 9 . A study analyzing the product of 6HLNO by nuclear magnetic resonance and continuous-flow mass spectrometry supports the flavin-dependent oxidation of the C-N bond of the pyrrolidone ring followed by non-enzymatic hydrolysis 10 . This mechanism is consistent with that generally accepted for flavin-dependent amine oxidases 14,15 .…”