Three nicotine demethylase genes mediate nornicotine biosynthesis in Nicotiana tabacum L.: Functional characterization of the CYP82E10 gene

Lewis, Ramsey S.; Bowen, Steven W.; Keogh, Matthew R.; Dewey, Ralph E.

doi:10.1016/j.phytochem.2010.09.011

Cited by 72 publications

(87 citation statements)

References 30 publications

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“…CYP82E4, CYP82E5 and CYP82E10 encode functional nicotine N-demethylases (Siminszky et al, 2005, Lewis et al, 2010 whereas two others CYP82E2…”

Section: Cytochrome P450s In Alkaloid Biosynthesismentioning

confidence: 99%

“…Since nicotine N-demethylase (NND), the main source for conversion of nicotine to nornicotine, is encoded by cytochrome P450 monooxygenase genes of the CYP82E subfamily in N. tabacum (Lewis et al, 2010, Siminszky et al, 2005, PCR using CYP82E specific primers based on the sequences of the conserved regions of the subfamily in N. tabacum (Table 3-1) was optimised to amplify the potential loci in Australian Nicotiana species. Based on the agarose gel electrophoresis results, genomic DNA corresponding to the conserved region of CYP82E related genes have been amplified in all of the studied Australian Nicotiana species (Figure 3-1).…”

Section: Cyp82e Genes In Australian Nicotiana Sppmentioning

confidence: 99%

“…Instead, the functionality of the loci has been compared between a few selected high converters and low or nonconverter species. The functional CYP82E loci, especially E4, have been reported to be triggered by curing which leads to a high level of transcript accumulation only in high converter species, while the transcript accumulation happens in very low and negligible levels in low or nonconverters (Lewis et al, 2010). This could be an explanation for the observed significantly elevated levels of transcripts (3 to 4 fold) in the cured leaves of only high converter species N. goodspeedii, N. cavicola and N. velutina, while no significant difference was found between the level of transcript accumulation in fresh leaves with the fresh and cured leaves of the low or nonconverter species N. gossei and N.…”

Section: Megalosiphon N Simulans N Occidentalis Ssp Occidentalismentioning

confidence: 99%

“…In human liver microsomes, nicotine Ndemethylation is catalysed by the P450 subfamilies CYP2A and CYP2B (Yamanaka et al, 2005). There are at least five CYP82E related genes in N. tabacum: CYP82E4, CYP82E5 and CYP82E10 encode functional nicotine N-demethylases (Siminszky et al, 2005, Lewis et al, 2010 whereas two others CYP82E2 and CYP82E3 encode inactive enzymes (Chakrabarti et al, 2007, Gavilano and. Investigations conducted on conversion locus analysis in Nicotiana spp.…”

Section: Introductionmentioning

confidence: 99%

“…Investigations conducted on conversion locus analysis in Nicotiana spp. are limited to the varieties of the important commercial tobacco, mainly N. tabacum (Cai et al, 2012a, Siminszky et al, 2005, Chakrabarti et al, 2008, Gavilano et al, 2006, Lewis et al, 2010) and its progenitor species N. tomentosiformis (Mann et al, 1964), and N. sylvestris (Chakrabarti et al, 2007). There is also a report of the loci and their functionality in a few South American wild species including N. langsdorffii and N. alata (Pakdeechanuan et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

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Investigations into the pituri plant: nicotine content, nicotine conversion to nornicotine, nicotine release and cytotoxicity of Australian native Nicotiana spp.

Moghbel¹

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The Aboriginal population of Central Australia use endemic Nicotiana spp. to make a smokeless tobacco product known as pituri that they chew/suck for nicotine absorption. This thesis describes the relative abundance of nicotine alkaloids amongst Australian Nicotiana spp., with special focus on the molecular characteristics of nicotine to nornicotine conversion.The most popular chewed species, N. gossei, is investigated for nicotine release and cytotoxicity in comparison to similar products to gain insight into potential hazards to pituri users.To analyse the alkaloids of Nicotiana leaves, a HPLC-UV method was developed to separate and quantify six closely related alkaloids (nicotine, nornicotine, anatabine, anabasine, myosmine, cotinine). A C18 column with a mobile phase of ammonium formate buffer (pH 10.5) separated the six alkaloids within 13 min with detection at 260 nm. Linearity, precision and reproducibility were satisfactory. The limit of quantification was 2.8 and 4.8 µg/mL for nornicotine and nicotine, respectively, and below 2 µg/mL for other alkaloids. This method quantifies more alkaloids and in less time than previously reported methods.Tobacco alkaloids are responsible in formation of carcinogenic tobacco specific nitrosamines such as N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). To quantify NNN and NNK, a fast LC-MS/MS method was developed using a HILIC column with a triple quadrupole tandem mass spectrometry. The linearity, accuracy, recovery and repeatability for this method were satisfactory, with quantification limit of 2.6 and 4.3 ng/mL for NNN and NNK, respectively. Nornicotine results from demethylation of nicotine, and is associated with negative effects on health. A group of cytochrome P450 genes that are mainly expressed during senescence or drying is involved in this conversion. The conversion loci were amplified in all studied 24 taxa, and sequenced in 6 selected species that contrast in conversion phenotype. Transcript accumulation of the responsible loci in fresh versus dried leaves of low or nonconverter N. gossei, N. excelsior and N. benthamiana maintained a steady level or a slight increase, but increased by 3 fold in cured leaves of the high converter N. goodspeedii, N. velutina and N. cavicola. This indicates the presence of functional loci that are triggered by curing only in high ii converter species and poses a potential risk for chewers of these species due to their greater potential for nornicotine production.The release of nicotine from the leaves of N. gossei was compared to that from a Swedish snus, the CORESTA reference smokeless tobacco (CRP2), and Nicabate chewing gum. A model buccal cavity system was developed and three different chewing conditions, performed manually were tested over 120 minutes: no chewing action, initial chewing and chewing at 15-minute intervals. To simulate the effect of alkaline wood ash, the effect of alkaline pH on the release of nicotine from N. gossei dry leaves was also evaluated. Samples ...

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“…CYP82E4, CYP82E5 and CYP82E10 encode functional nicotine N-demethylases (Siminszky et al, 2005, Lewis et al, 2010 whereas two others CYP82E2…”

Section: Cytochrome P450s In Alkaloid Biosynthesismentioning

confidence: 99%

Section: Cyp82e Genes In Australian Nicotiana Sppmentioning

confidence: 99%

Section: Megalosiphon N Simulans N Occidentalis Ssp Occidentalismentioning

confidence: 99%