Theoretical study on the N-demethylation mechanism of theobromine catalyzed by P450 isoenzyme 1A2

Jing, Tao; Kou, Yuan; Xue, Zhiyu; Wang, Yongting; Zhang, Yan; Qiu, Chen; Chen, Zeqin; Yang, Xue

doi:10.1016/j.jmgm.2015.06.017

Cited by 13 publications

(5 citation statements)

References 36 publications

(39 reference statements)

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“…The potential of 3′ as the direct substrate for this transformation is supported by two criteria: (a) the intramolecular hydrogen bond between the formamide oxygen and the hemiaminal proton of 3′ would make the formamide carbon a better electrophile, and thus more susceptible to hydrolysis, and (b) the subsequent deformylated hemiaminal ( 3′′ ) is a stronger base than 3′ and could therefore more readily promote decomposition into 5 (Figure S7). To test this hypothesis, we prepared in vitro reactions with both Gs CYP71DA12-enriched microsomes and the previously purified Gs ABH1 enzyme. When 3 and NADPH were included in the reaction, we observed not only the accumulation of 3′ and 4a , but also Gs ABH1-dependent accumulation of 5 (Figure S7).…”

Section: Resultsmentioning

confidence: 99%

Total Biosynthesis of the Tubulin-Binding Alkaloid Colchicine

Nett

Sattely

2021

J. Am. Chem. Soc.

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Colchicine (1) is a bioactive plant alkaloid from Colchicum and Gloriosa species that is used as a pharmaceutical treatment for inflammatory diseases, including gouty arthritis and familial Mediterranean fever. The activity of this alkaloid is attributed to its ability to bind tubulin dimers and inhibit microtubule assembly, which not only promotes anti-inflammatory effects, but also makes colchicine a potent mitotic poison. The biochemical origins of colchicine biosynthesis have been investigated for over 50 years, but only recently has the underlying enzymatic machinery become clear. Here, we report the discovery of multiple pathway enzymes from Gloriosa superba that allows for the reconstitution of a complete metabolic route to 1. This includes three enzymes that process a previously established tropolone-containing intermediate into 1 via tailoring of the nitrogen atom. We further demonstrate the total biosynthesis of enantiopure (−)-1 from primary metabolites via heterologous production in a model plant, thus enabling future efforts for the metabolic engineering of this medicinal alkaloid. Additionally, our results provide insight into the timing and tissue specificity for the late stage modifications required in colchicine biosynthesis, which are likely connected to the biological functions for this class of medicinal alkaloids in native producing plants.

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Section: Resultsmentioning

confidence: 99%

Total Biosynthesis of the Tubulin-Binding Alkaloid Colchicine

Nett

Sattely

2021

J. Am. Chem. Soc.

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“…CYP392A16 metabolized 21% of the pyflubumide carboxamide NNI-0711-NH (with a concentration of 25 μM) after a 1-hour incubation. HPLC-MS/MS analysis of the reaction mixtures pointed to N-demethylation as the likely mechanism of the CYP392A16 reaction (Fig 7) [46]. Demethylation is expected to result in a mass difference of -14 Da between the NNI-0711-NH and reaction metabolite product ions.…”

Section: Cyp392a16 Demethylates the Toxic Pyflubumide Carboxamidementioning

confidence: 99%

High-resolution genetic mapping reveals cis-regulatory and copy number variation in loci associated with cytochrome P450-mediated detoxification in a generalist arthropod pest

et al. 2021

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Chemical control strategies are driving the evolution of pesticide resistance in pest populations. Understanding the genetic mechanisms of these evolutionary processes is of crucial importance to develop sustainable resistance management strategies. The acaricide pyflubumide is one of the most recently developed mitochondrial complex II inhibitors with a new mode of action that specifically targets spider mite pests. In this study, we characterize the molecular basis of pyflubumide resistance in a highly resistant population of the spider mite Tetranychus urticae. Classical genetic crosses indicated that pyflubumide resistance was incompletely recessive and controlled by more than one gene. To identify resistance loci, we crossed the resistant population to a highly susceptible T. urticae inbred strain and propagated resulting populations with and without pyflubumide exposure for multiple generations in an experimental evolution set-up. High-resolution genetic mapping by a bulked segregant analysis approach led to the identification of three quantitative trait loci (QTL) linked to pyflubumide resistance. Two QTLs were found on the first chromosome and centered on the cytochrome P450 CYP392A16 and a cluster of CYP392E6-8 genes. Comparative transcriptomics revealed a consistent overexpression of CYP392A16 and CYP392E8 in the experimental populations that were selected for pyflubumide resistance. We further corroborated the involvement of CYP392A16 in resistance by in vitro functional expression and metabolism studies. Collectively, these experiments uncovered that CYP392A16 N-demethylates the toxic carboxamide form of pyflubumide to a non-toxic compound. A third QTL coincided with cytochrome P450 reductase (CPR), a vital component of cytochrome P450 metabolism. We show here that the resistant population harbors three gene copies of CPR and that this copy number variation is associated with higher mRNA abundance. Together, we provide evidence for detoxification of pyflubumide by cytochrome P450s that is likely synergized by gene amplification of CPR.

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“…CYP392A16 metabolized 21% of the pyflubumide carboxamide NNI-0711-NH (with a concentration of 25 µM) after a 1-hour incubation. HPLC-MS/MS analysis of the reaction mixtures pointed to N-demethylation as the likely mechanism of the CYP392A16 reaction (Fig 7) [44]. Demethylation is expected to result in a mass difference of -14 Da between the NNI-0711-NH and reaction metabolite product ions.…”

Section: Resultsmentioning

confidence: 99%

High-resolution genetic mapping revealscis-regulatory and copy number variation in loci associated with cytochrome P450-mediated detoxification in a generalist arthropod pest

Fotoukkiaii

Wybouw

Kurlovs

et al. 2021

Preprint

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Chemical control strategies are driving the evolution of pesticide resistance in pest populations. Understanding the genetic mechanisms of these evolutionary processes is of crucial importance to develop sustainable resistance management strategies. The acaricide pyflubumide is one of the most recently developed mitochondrial complex II inhibitors with a new mode of action that specifically targets spider mite pests. In this study, we characterize the molecular basis of pyflubumide resistance in a highly resistant population of the spider mite Tetranychus urticae . Classical genetic crosses indicated that pyflubumide resistance was incompletely recessive and controlled by more than one gene. To identify resistance loci, we crossed the resistant population to a highly susceptible T. urticae inbred strain and propagated resulting populations with and without pyflubumide exposure for multiple generations in an experimental evolution set-up. High-resolution genetic mapping by a bulked segregant analysis approach led to the identification of three quantitative trait loci (QTL) linked to pyflubumide resistance. Two QTLs were found on the first chromosome and centered on the cytochrome P450 CYP392A16 and a cluster of CYP392E6-8 genes. Comparative transcriptomics revealed a consistent overexpression of CYP392A16 and CYP392E8 in the experimental populations that were selected for pyflubumide resistance. We further corroborated the involvement of CYP392A16 in resistance by in vitro functional expression and metabolism studies. Collectively, these experiments uncovered that CYP392A16 N-demethylates the toxic carboxamide form of pyflubumide to a non-toxic compound . A third QTL coincided with cytochrome P450 reductase ( CPR ), a vital component of cytochrome P450 metabolism. We show here that the resistant population harbors three gene copies of CPR and that this copy number variation is associated with higher mRNA abundance . Together, we provide evidence for detoxification of pyflubumide by cytochrome P450s that is likely synergized by gene amplification of CPR .

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Theoretical study on the N-demethylation mechanism of theobromine catalyzed by P450 isoenzyme 1A2

Cited by 13 publications

References 36 publications

Total Biosynthesis of the Tubulin-Binding Alkaloid Colchicine

Total Biosynthesis of the Tubulin-Binding Alkaloid Colchicine

High-resolution genetic mapping reveals cis-regulatory and copy number variation in loci associated with cytochrome P450-mediated detoxification in a generalist arthropod pest

High-resolution genetic mapping revealscis-regulatory and copy number variation in loci associated with cytochrome P450-mediated detoxification in a generalist arthropod pest

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