Nicotine-N′-Oxidation by Flavin Monooxygenase Enzymes

Abstract

Background: The major mode of metabolism of nicotine is by hydroxylation via cytochrome P450 (CYP) 2A6, but it can also undergo glucuronidation by UDP-glucuronosyltransferases and oxidation by flavin monooxygenases (FMO). The goal of this study was to examine the potential importance of FMOs in nicotine metabolism and assess the potential impact of missense polymorphisms in active FMOs on nicotine-N 0oxide (NOX) formation. Methods: Urine samples from 106 current Chinese smokers were analyzed for nicotine metabolites by mass spectrometry. Wild-type FMOs 1-5 and their most prevalent nonsynonymous variants were cloned and overexpressed in HEK293 cells, and were tested in oxidation reactions against nicotine. Results: A strong inverse correlation was observed between the ratio of urinary 3 0-hydroxycotinine/cotinine, a measure of CYP2A6 activity, and the urinary levels of NOX alone (r ¼ À0.383; P < 0.001) or NOX measured as a ratio of total nicotine metabolites (r ¼ À0.414; P < 0.001) in smokers. In addition to FMO1 and FMO3, the functional FMO2 427Q isoform was active against nicotine, whereas FMO4 and FMO5 exhibited low activity against nicotine (K m > 5.0 mmol/L). Significant (P < 0.05) decreases in N 0-oxidation activity (V max /K m) were observed for the FMO1 I303V , FMO3 N61S , FMO3 D132H , FMO3 V257M , and FMO3 E308G variants in vitro when compared with their respective wild-type isoforms; the truncated FMO2 Q472stop isoform exhibited no enzyme activity. Conclusions: These data indicate that increases in nicotine-N 0-oxidation occur in subjects with deficient CYP2A6 activity, and that several FMO enzymes are active in nicotine-N 0oxidation. Impact: Several common missense FMO variants are associated with altered enzyme activity against nicotine and may play an important role in nicotine metabolism in low-CYP2A6 activity subjects.

Flavin-dependent N-hydroxylating enzymes: distribution and application

“…Recently, hFMO3 was reported to exhibit activity towards nicotine (Fig. 5c ) (Perez-Paramo et al 2019 ). The substrate versatility of this enzyme makes it an attractive catalyst for future applications with complex substrates and towards the synthesis of drug metabolites.…”

“…As already introduced above, these FMOs belong to the same group of flavoprotein monooxygenases and are able to oxygenate carbon-linked heteroatoms (van Berkel et al 2006 ). The five hFMO enzymes exhibit a tissue-specific expression pattern in adults (Perez-Paramo et al 2019 ): hFMO1 is expressed in kidneys and hFMO3–5 in the liver, whereas hFMO2 is mainly expressed in the lungs. hFMO5, which was shown to catalyze Baeyer-Villiger oxidation reactions (Fiorentini et al 2016 ; Fiorentini et al 2017 ), is now commercially available (Gecco Biotech B.V.).…”

Genetic variants in CYP2A6 and UGT1A9 genes associated with urinary nicotine metabolites in young Mexican smokers

“…Genomic DNA was extracted with the TSNT technique (lysis buffer: 2% Triton, 1% SDS, Fig. 1 Schematic of major nicotine metabolites (adapted from Perez-Paramo et al [7]).…”

“…Enzymes of nicotine metabolism are highly polymorphic and their genetic variants can cause differences in enzyme activity [7,[10][11][12][13][14][15][16]. This may translate into inter-individual differences in the concentration of nicotine in the body and to differences in the number of cigarettes consumed by smokers [17].…”

“…1 ), resulting in more polar molecules that are easily excreted in the urine [ 5 , 6 ]. Also, nicotine and cotinine are oxidized to nicotine- N ′-Oxide and cotinine- N -Oxide by flavin monooxygenases enzymes (FMOs) [ 7 ], and it is believed that 4-oxo-4-(3-pyridyl) butanoic acid (4HPBA) is formed by the hydroxylation of nicotine by the CYP2A6 enzyme [ 8 , 9 ].

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