Investigation of the mechanism of nicotine demethylation in Nicotiana through 2H and 15N heavy isotope effects: Implication of cytochrome P450 oxidase and hydroxyl ion transfer

Molinié, Roland; Kwiecień, Renata A.; Paneth, Piotr; Hatton, Wilfried; Lebreton, Jacques; Robins, Richard J.

doi:10.1016/j.abb.2006.12.013

Cited by 12 publications

(14 citation statements)

References 47 publications

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“…It was, therefore, our objective to investigate whether also isotope effects can be indicative of the prevailing transformation mechanism as brought forward in previous studies. [44][45][46][47] For SET an inverse N isotope effect (depletion of the 15 N-isotopologue in the residual substance pool) was observed by Skarpeli-Liati et al 48 in oxidation of substituted anilines by manganese oxide. Using computational calculations, we aimed to investigate whether a similar inverse isotope effect is predicted for atrazine, and whether the opposite patterna normal secondary N-isotope effect (enrichment of the 15 N-isotopologue in the residual substance pool)would be expected for HAT.…”

Section: Introductionmentioning

confidence: 92%

Cytochrome P450-catalyzed dealkylation of atrazine byRhodococcussp. strain NI86/21 involves hydrogen atom transfer rather than single electron transfer

et al. 2014

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Cytochrome P450 enzymes are responsible for a multitude of natural transformation reactions. For oxidative N-dealkylation, single electron (SET) and hydrogen atom abstraction (HAT) have been debated as underlying mechanisms. Combined evidence from (i) product distribution and (ii) isotope effects indicate that HAT, rather than SET, initiates N-dealkylation of atrazine to desethyl- and desisopropylatrazine by the microorganism Rhodococcus sp. strain NI86/21. (i) Product analysis revealed a non-selective oxidation at both the αC and βC-atom of the alkyl chain, which is expected for a radical reaction, but not SET. (ii) Normal (13)C and (15)N as well as pronounced (2)H isotope effects (εcarbon: -4.0‰ ± 0.2‰; εnitrogen: -1.4‰ ± 0.3‰, KIEH: 3.6 ± 0.8) agree qualitatively with calculated values for HAT, whereas inverse (13)C and (15)N isotope effects are predicted for SET. Analogous results are observed with the Fe(iv)[double bond, length as m-dash]O model system [5,10,15,20-tetrakis(pentafluorophenyl)porphyrin-iron(iii)-chloride + NaIO4], but not with permanganate. These results emphasize the relevance of the HAT mechanism for N-dealkylation by P450.

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Section: Introductionmentioning

confidence: 92%

Cytochrome P450-catalyzed dealkylation of atrazine byRhodococcussp. strain NI86/21 involves hydrogen atom transfer rather than single electron transfer

et al. 2014

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“…28−30 When the reaction coordinate was calculated for the first time for nicotine metabolism using a small model semi ab initio method (SAM1), it was concluded that the oxygen rebound step was rate-controlling on the basis of the correlation between experimentally determined 2 H and 15 N heavy isotope effects and those calculated using this model. 31 However, although this was the first time that the reaction coordinate was obtained for nicotine demethylation, the influence of neither the protein environment nor the charge state of nicotine was taken into consideration.…”

Section: ■ Introductionmentioning

confidence: 99%

Cytochrome P450-Catalyzed Degradation of Nicotine: Fundamental Parameters Determining Hydroxylation by Cytochrome P450 2A6 at the 5′-Carbon or the N-Methyl Carbon

et al. 2012

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The oxidation of (2'S)-nicotine in the active site of human cytochrome P450 2A6 has been subjected to a detailed analysis by theoretical quantum mechanical/molecular mechanical (QM/MM) calculations linked with a theoretical and experimental study of the associated isotope effects. The study has focused on seeking an explanation as to why oxidation at the 5'-carbon position (A) is favored over oxidation at the methyl carbon (CMe) position (B). It is deduced that the choice of hydrogen for abstraction is not determined by geometric features of the active site, but by the lower energy barrier associated with 5' oxidation. N-Demethylation leading to N-hydroxymethylnornicotine requires ca. 6.5 kcal/mol more energy to transfer a hydrogen atom than is required for oxidation on the carbon 5'. Neither protonation of the pyrrolidine nitrogen (N1') nor inclusion of a water molecule in the reaction process influences the balance between the two oxidation pathways. In both cases, the hydrogen transfer step is rate limiting. An analysis of the calculated kinetic isotope effects indicates that the presence of a (2)H in either the C5' or the CMepositions has a significant effect on the reaction kinetics. However, the experimental values of around 2.2-2.6 are considerably lower than those predicted by theoretical calculations (9.3 and 6.9 for C5' or the CMe positions, respectively, in the LS state of Cpd I), typical of the masking commonly found for CYP450 reactions. The fact that similar values are found for cotinine formation from both substrates, however, may indicate that the measured value is not that for H-abstraction but, rather, is a combined value for (2)H influence on electronic redistribution between iminium states of the pyrrolidine ring. This is the first time that oxidation at the C5' or the CMe positions has been directly compared and that isotope effects have been obtained for this reaction in a human cytochrome P450 reaction.

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“…Rather, and as a general principle for studies of isotope effects during biotransformation, quantification is best performed directly on the culture medium. For compounds that contain a chromophore, such as nicotine, this is simply achieved by HPLC 9. However, for compounds less readily detected by absorption spectroscopy, such as tropine, quantitative 1 H NMR seems to offer a reliable means by which to quantify polar analytes.…”

Section: Discussionmentioning

confidence: 99%

“…As part of a study of the mechanisms of the degradation and/or biotransformation of plant‐derived alkaloids, we are measuring the 15 N kinetic isotope effects (KIEs) associated with degradative reactions 8. From experience,9 the magnitude of the isotope shift (Δ δ 15 N) a is only of a few δ 15 N (‰) units, making it particularly important to determine the values as accurately10 as possible and to ensure that there is no influence of the preparation procedure and analytical conditions used to make the measurements 11. Stable isotope ratio measurement (irm) in continuous flow with on‐line elemental analysis (EA) or gas chromatography (GC) interfaced with mass spectrometry (MS) is a powerful tool in this context.…”

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confidence: 99%

Determination of nitrogen‐15 isotope fractionation in tropine: evaluation of extraction protocols for isotope ratio measurement by isotope ratio mass spectrometry

Molinié

Kwiecień

Silvestre

et al. 2009

Rapid Comm Mass Spectrometry

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N-Demethylation of tropine is an important step in the degradation of this compound and related metabolites. With the purpose of understanding the reaction mechanism(s) involved, it is desirable to measure the 15N kinetic isotope effects (KIEs), which can be accessed through the 15N isotope shift (Deltadelta15N) during the reaction. To measure the isotope fractionation in 15N during tropine degradation necessitates the extraction of the residual substrate from dilute aqueous solution without introducing artefactual isotope fractionation. Three protocols have been compared for the extraction and measurement of the 15N/14N ratio of tropine from aqueous medium, involving liquid-liquid phase partitioning or silica-C18 solid-phase extraction. Quantification was by gas chromatography (GC) on the recovered organic phase and delta15N values were obtained by isotope ratio measurement mass spectrometry (irm-MS). Although all the protocols used can provide satisfactory data and both irm-EA-MS and irm-GC-MS can be used to obtain the delta15N values, the most convenient method is liquid-liquid extraction from a reduced aqueous volume combined with irm-GC-MS. The protocols are applied to the measurement of 15N isotope shifts during growth of a Pseudomonas strain that uses tropane alkaloids as sole source of carbon and nitrogen. The accuracy of the determination of the 15N/14N ratio is sufficient to be used for the determination of 15N-KIEs.

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Investigation of the mechanism of nicotine demethylation in Nicotiana through 2H and 15N heavy isotope effects: Implication of cytochrome P450 oxidase and hydroxyl ion transfer

Cited by 12 publications

References 47 publications

Cytochrome P450-catalyzed dealkylation of atrazine byRhodococcussp. strain NI86/21 involves hydrogen atom transfer rather than single electron transfer

Cytochrome P450-catalyzed dealkylation of atrazine byRhodococcussp. strain NI86/21 involves hydrogen atom transfer rather than single electron transfer

Cytochrome P450-Catalyzed Degradation of Nicotine: Fundamental Parameters Determining Hydroxylation by Cytochrome P450 2A6 at the 5′-Carbon or the N-Methyl Carbon

Determination of nitrogen‐15 isotope fractionation in tropine: evaluation of extraction protocols for isotope ratio measurement by isotope ratio mass spectrometry

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