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Cited by 12Plant biochemistry and biosynthesisPlant tissue culture and regenerationPlant Gene Expression Analysis

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Nicotine biosynthesis is completed by cryptic activating glucosylation

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Schwabe, Benjamin T. W., Angstman, Isabelle M., Vollheyde, Katharina, Ingold, Zoe, Li, Jiacheng, Stankevich, Ksenia S., Spicer, Christopher D., Fascione, Martin A., Grogan, Gideon, Geu-Flores, Fernando, Lichman, Benjamin R.·2026·Nature Communications
DOI 10.1038/s41467-026-72705-0PMC PMC13184244

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Abstract

Nicotine is a neuroactive alkaloid produced by tobacco ( Nicotiana tabacum ) as a defence against herbivory, and an addictive stimulant that has been used by humans for millennia. Despite its significance, the core steps of its biosynthesis have remained elusive. Here, we demonstrate the in vitro reconstruction of a four-enzyme stereoselective biocatalytic cascade that forms ( S )-nicotine from nicotinic acid and N -methylpyrrolinium. This cascade includes two glucose-processing enzymes that participate in a cryptic activating glucosylation step. We also reconstruct this pathway in planta and present high resolution X-ray structures of the key carbon-carbon bond forming reductase-oxidase pair bound to their substrate and product, respectively. This work establishes the complete biosynthetic pathway to nicotine, providing new gene targets for controlling alkaloid production in Nicotiana and unlocking enzymatic routes to pyridine alkaloids.

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Fig. 1 Proposed biosynthesis of nicotine. Hypothesized biosynthetic route to (S)-nicotine ((S)-1), showing origins of the pyridine (blue) and N-methylpyrrolidine (purple) rings. The Mannich-like scaffold formation is proposed to proceed via a multi-step ‘nicotine synthase’ cascade16. Compounds that accumulate in Nicotiana silencing/knock-out experiments are depicted. The red hydrogens follow the fate of the hydride transferred via enzymatic reduction of 2.
Fig. 1 Proposed biosynthesis of nicotine. Hypothesized biosynthetic route to (S)-nicotine ((S)-1), showing origins of the pyridine (blue) and N-methylpyrrolidine (purple) rings. The Mannich-like scaffold formation is proposed to proceed via a multi-step ‘nicotine synthase’ cascade16. Compounds that accumulate in Nicotiana silencing/knock-out experiments are depicted. The red hydrogens follow the fate of the hydride transferred via enzymatic reduction of 2.CC BY · PubMed Central
Fig. 2 Identification of biosynthetic gene clusters and gene candidates. A Homeologous biosynthetic gene clusters identified in the N. tabacum 2017 genome assembly37 consisting of β-glucosidase (β-GD, blue), reductase (A622, red) and transporter (MATE, yellow) genes. Scaffold/chromosome number is described above the linear genome strand, with numbers at either end representing nucleotide position. Scaffold 884 is unplaced in this assembly but in the Sierro et al. 2024 assembly it is part of chromosome 1675. The gene model between A622 and MATE1 in scaffold 884 (884g0040) encodes a protein that contains a domain with homology to a reverse transcriptase domain (PANTHER ID PTHR33116). B Heatmap of expression of selected nicotine genes, including β-GD and UGT candidates across tissue types. Average expression calculated in each tissue type and values normalized per gene. Number of samples in each category above each column. C Clustered heatmap showing all-by-all correlations (PCC) of nicotine biosynthesis and related genes across 824 samples. TPM values obtained from PEO40. D Root expression levels of selected nicotine biosynthesis genes and UGT candidates following topping treatment, and their correlation (PCC) with A622 responses. Data obtained from Qin et al. 202041. Details of genes available in Supplementary Data 1.
Fig. 2 Identification of biosynthetic gene clusters and gene candidates. A Homeologous biosynthetic gene clusters identified in the N. tabacum 2017 genome assembly37 consisting of β-glucosidase (β-GD, blue), reductase (A622, red) and transporter (MATE, yellow) genes. Scaffold/chromosome number is described above the linear genome strand, with numbers at either end representing nucleotide position. Scaffold 884 is unplaced in this assembly but in the Sierro et al. 2024 assembly it is part of chromosome 1675. The gene model between A622 and MATE1 in scaffold 884 (884g0040) encodes a protein that contains a domain with homology to a reverse transcriptase domain (PANTHER ID PTHR33116). B Heatmap of expression of selected nicotine genes, including β-GD and UGT candidates across tissue types. Average expression calculated in each tissue type and values normalized per gene. Number of samples in each category above each column. C Clustered heatmap showing all-by-all correlations (PCC) of nicotine biosynthesis and related genes across 824 samples. TPM values obtained from PEO40. D Root expression levels of selected nicotine biosynthesis genes and UGT candidates following topping treatment, and their correlation (PCC) with A622 responses. Data obtained from Qin et al. 202041. Details of genes available in Supplementary Data 1.CC BY · PubMed Central
Fig. 3 In vitro reconstitution of nicotine biosynthesis. A Proposed chemical transformations in nicotine biosynthesis reconstitution. B Products of in vitro cascades. Each row corresponds to an in vitro reaction, with the matrix showing presence/absence (blue/white) of reaction components, alongside UDP-Glc and NADPH. Each column shows extracted ion chromatograms (EICs, m/z ± 0.15). Blue chromatograms are chemically verified standards, scaled for clarity. Red chromatograms highlight key products. MS2 data is available in Supplementary Fig. 3. C Quantification of in vitro cascades. Each row of bars corresponds to an in vitro reaction, with the matrix showing presence/absence (blue/white) of reaction components. Each column of bars shows the peak area of EICs (m/z ± 0.15) corresponding to the masses of the chemical depicted above (left-to-right: 10, 12, 13, 1). Compound identity was validated by standard retention time, and MS2 spectra (Supplementary Fig. 3). Red bars highlight key products. The letters above bars are significant groupings of peak area, determined per chemical (p < 0.05, Tukey post-hoc test, see Supplementary Data 5). The concentration of nicotine produced (from 1 mM of 2) was determined via an external standard curve; error bars show standard error (inset). Bars show mean EIC peak area (n = 3); points are individual replicates; error bars show standard deviation. Source data are provided as a Source Data file.
Fig. 3 In vitro reconstitution of nicotine biosynthesis. A Proposed chemical transformations in nicotine biosynthesis reconstitution. B Products of in vitro cascades. Each row corresponds to an in vitro reaction, with the matrix showing presence/absence (blue/white) of reaction components, alongside UDP-Glc and NADPH. Each column shows extracted ion chromatograms (EICs, m/z ± 0.15). Blue chromatograms are chemically verified standards, scaled for clarity. Red chromatograms highlight key products. MS2 data is available in Supplementary Fig. 3. C Quantification of in vitro cascades. Each row of bars corresponds to an in vitro reaction, with the matrix showing presence/absence (blue/white) of reaction components. Each column of bars shows the peak area of EICs (m/z ± 0.15) corresponding to the masses of the chemical depicted above (left-to-right: 10, 12, 13, 1). Compound identity was validated by standard retention time, and MS2 spectra (Supplementary Fig. 3). Red bars highlight key products. The letters above bars are significant groupings of peak area, determined per chemical (p < 0.05, Tukey post-hoc test, see Supplementary Data 5). The concentration of nicotine produced (from 1 mM of 2) was determined via an external standard curve; error bars show standard error (inset). Bars show mean EIC peak area (n = 3); points are individual replicates; error bars show standard deviation. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 4 Stereoselectivity and substrate scope of nicotine biosynthetic cascade. A Stereoselective formation of (S)-nicotine (1). Nicotine formation was achieved through one-pot enzymatic cascade (2, 3, NaGT, NaGR, NicGH) with and without NicGS detected by chiral HPLC (260 nm). Signal intensity of peaks and standards (blue) was scaled for comparison. B Induction of stereoselectivity by NicGS. EIC (m/z 325.2 ± 0.15) showing (R, S)-nicotine glucoside (13) formation from 2 with NaGT, NaGR and NMP, with or without NicGS. Chemically verified standards are blue. Signal intensity was scaled for comparison. C Formation of nornicotine (16) and D anabasine (6) via the nicotine biosynthetic cascade (2, NaGT, NaGR, NicGS, NicGH) with 15 or 8, respectively, either synthesized or formed enzymatically by OLADO.
Fig. 4 Stereoselectivity and substrate scope of nicotine biosynthetic cascade. A Stereoselective formation of (S)-nicotine (1). Nicotine formation was achieved through one-pot enzymatic cascade (2, 3, NaGT, NaGR, NicGH) with and without NicGS detected by chiral HPLC (260 nm). Signal intensity of peaks and standards (blue) was scaled for comparison. B Induction of stereoselectivity by NicGS. EIC (m/z 325.2 ± 0.15) showing (R, S)-nicotine glucoside (13) formation from 2 with NaGT, NaGR and NMP, with or without NicGS. Chemically verified standards are blue. Signal intensity was scaled for comparison. C Formation of nornicotine (16) and D anabasine (6) via the nicotine biosynthetic cascade (2, NaGT, NaGR, NicGS, NicGH) with 15 or 8, respectively, either synthesized or formed enzymatically by OLADO.CC BY · PubMed Central
Fig. 5 In vitro enzymatic cascade with labelled nicotinic acid. A Schematic of reaction cascade with nicotinic acid-(ring-d4) (2-d4). B Selected in vitro cascades with 2-d4 substrate. Bar charts show EIC peak areas of product (from L-to-R: 10, 12, 13, 1) isotopologues (all m/z ± 0.15 except for d3-12 which was m/z ± 0.5 due to peak shape distortion). Reactions performed in triplicate, bars show mean peak area, points show individual measurements, and error bars show standard deviation. Each row corresponds to an in vitro reaction, with the matrix showing presence/absence (blue/white) of reaction components. Letters above bars are significant groupings of d3/d4 isotopologue ratios, determined per chemical across all reaction combinations (p < 0.05, Tukey’s HSD test, Supplementary Data 6, see Supplementary Fig. 8 for all reactions). C–E Conversion of nicotinic acid (2) isotopologues by the nicotine biosynthetic cascade (NaGT, NaGR, NicGH, NADPH and UDP-Glc) with and without NicGS. Bar charts show peak areas of products from EICs. Reactions were performed in triplicate (points); bars show mean peak area; error bars show standard deviation. Source data are provided as a Source Data file.
Fig. 5 In vitro enzymatic cascade with labelled nicotinic acid. A Schematic of reaction cascade with nicotinic acid-(ring-d4) (2-d4). B Selected in vitro cascades with 2-d4 substrate. Bar charts show EIC peak areas of product (from L-to-R: 10, 12, 13, 1) isotopologues (all m/z ± 0.15 except for d3-12 which was m/z ± 0.5 due to peak shape distortion). Reactions performed in triplicate, bars show mean peak area, points show individual measurements, and error bars show standard deviation. Each row corresponds to an in vitro reaction, with the matrix showing presence/absence (blue/white) of reaction components. Letters above bars are significant groupings of d3/d4 isotopologue ratios, determined per chemical across all reaction combinations (p < 0.05, Tukey’s HSD test, Supplementary Data 6, see Supplementary Fig. 8 for all reactions). C–E Conversion of nicotinic acid (2) isotopologues by the nicotine biosynthetic cascade (NaGT, NaGR, NicGH, NADPH and UDP-Glc) with and without NicGS. Bar charts show peak areas of products from EICs. Reactions were performed in triplicate (points); bars show mean peak area; error bars show standard deviation. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 6 X-ray crystal structures of NaGR (A622) and NicGS (BBLa). A The crystal structure of NaGR bound to NADP+ and nicotinic acid N-glucoside (green net shows unrefined ligand density Fo-Fc at 3 σ). B Proposed mechanism of NaGR: NADPH reduces nicotinic acid N-glucoside-d6 on the re-face, adding a hydride in the pro-R position. C The crystal structure of NicGS with covalently bound FAD and ligand (S)-nicotine glucoside (green net shows unrefined ligand density Fo-Fc at 3 σ). D Proposed mechanism of NicGS, abstracting the pro-S hydride from dihydronicotine glucoside-d6 to yield nicotine glucoside.
Fig. 6 X-ray crystal structures of NaGR (A622) and NicGS (BBLa). A The crystal structure of NaGR bound to NADP+ and nicotinic acid N-glucoside (green net shows unrefined ligand density Fo-Fc at 3 σ). B Proposed mechanism of NaGR: NADPH reduces nicotinic acid N-glucoside-d6 on the re-face, adding a hydride in the pro-R position. C The crystal structure of NicGS with covalently bound FAD and ligand (S)-nicotine glucoside (green net shows unrefined ligand density Fo-Fc at 3 σ). D Proposed mechanism of NicGS, abstracting the pro-S hydride from dihydronicotine glucoside-d6 to yield nicotine glucoside.CC BY · PubMed Central
Fig. 7 In planta reconstitution of nicotine biosynthesis. Leaves of N. benthamiana were agroinfiltrated with a combination of nicotine biosynthesis genes (day 0), fed with nicotinamide-d4 (ND-d4) at day 4, and harvested for metabolite analysis at day 6. The gene combination producing the N-methylpyrrolinium (ODC, PMT and MPO) is abbreviated as NMP. A Accumulation of the labelled compounds is indicated at the top (d3 and d4 versions together). Each row corresponds to the gene combination shown in the matrix on the left (green/white indicates presence/absence). Traces are representative extracted ion chromatograms (EICs, m/z ± 0.005) corresponding to the labelled compounds (black) in comparison to chemically synthesized or commercial standards (blue) (see Supplementary Fig. 12 for MS2 spectra). Signal intensities are normalized to the largest peak on each column; standards were rescaled to match the largest peak. The nicotine and anabasine standards were analysed as a mixed sample. The insert on the right shows the chiral analysis of nicotine in the presence of all genes and in the NicGS drop-out (D.O.). B Individual quantification of d3 and d4 isotopologues corresponding to the compounds and gene combinations shown in (A). Bars show mean EIC peak area (n = 8); points are individual replicates; error bars show standard deviation (see statistics in Supplementary Data 11). Source data are provided as a Source Data file.
Fig. 7 In planta reconstitution of nicotine biosynthesis. Leaves of N. benthamiana were agroinfiltrated with a combination of nicotine biosynthesis genes (day 0), fed with nicotinamide-d4 (ND-d4) at day 4, and harvested for metabolite analysis at day 6. The gene combination producing the N-methylpyrrolinium (ODC, PMT and MPO) is abbreviated as NMP. A Accumulation of the labelled compounds is indicated at the top (d3 and d4 versions together). Each row corresponds to the gene combination shown in the matrix on the left (green/white indicates presence/absence). Traces are representative extracted ion chromatograms (EICs, m/z ± 0.005) corresponding to the labelled compounds (black) in comparison to chemically synthesized or commercial standards (blue) (see Supplementary Fig. 12 for MS2 spectra). Signal intensities are normalized to the largest peak on each column; standards were rescaled to match the largest peak. The nicotine and anabasine standards were analysed as a mixed sample. The insert on the right shows the chiral analysis of nicotine in the presence of all genes and in the NicGS drop-out (D.O.). B Individual quantification of d3 and d4 isotopologues corresponding to the compounds and gene combinations shown in (A). Bars show mean EIC peak area (n = 8); points are individual replicates; error bars show standard deviation (see statistics in Supplementary Data 11). Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 8 Identification of pathway intermediates and shunt products in Nicotiana roots. A Metabolite analysis of roots from N. benthamiana wild type, ΔNaGR29 and ΔNicGS31 lines (black) in comparison to standards (blue) (see MS2 verification in Supplementary Fig. 13). Traces are representative extracted ion chromatograms (EICs, m/z ± 0.005) and were normalized to the largest peak in each column; standards were re-scaled to match the largest peak. B Metabolite analysis of roots from N. tabacum var Samsun (black), compared to standards (blue) (see MS2 verification in Supplementary Fig. 14). The red trace is root extract sample spiked with 10 μM nicotine N-glucoside (13) to verify peak identity. Traces are representative EICs (m/z ± 005); standards were re-scaled to match the corresponding peaks in the N. tabacum samples.
Fig. 8 Identification of pathway intermediates and shunt products in Nicotiana roots. A Metabolite analysis of roots from N. benthamiana wild type, ΔNaGR29 and ΔNicGS31 lines (black) in comparison to standards (blue) (see MS2 verification in Supplementary Fig. 13). Traces are representative extracted ion chromatograms (EICs, m/z ± 0.005) and were normalized to the largest peak in each column; standards were re-scaled to match the largest peak. B Metabolite analysis of roots from N. tabacum var Samsun (black), compared to standards (blue) (see MS2 verification in Supplementary Fig. 14). The red trace is root extract sample spiked with 10 μM nicotine N-glucoside (13) to verify peak identity. Traces are representative EICs (m/z ± 005); standards were re-scaled to match the corresponding peaks in the N. tabacum samples.CC BY · PubMed Central
Fig. 9 Putative activities of NicGS (BBL). Possible mechanistic routes towards (S)−1 from 10. The blue background shows key NaGR and NicGH reactions and major products. The grey background shows possible reactions that are either non-enzymatically or catalysed by NicGS. Only compounds with blue or white background have been directly detected. To induce the stereoselective outcome, NicGS may operate through: dynamic kinetic resolution (catalysing just purple arrows and allowing other intermediates to non-enzymatically equilibrate), epimerisation of 5b via a ring opened intermediate (catalysing red and purple arrows), catalysis of the stereoselective Mannich reaction between 3 and 10 prior to oxidation, reversal of the non-desired Mannich reaction (opening (R)-5b into 3 and 10). Blue arrows show the predicted mechanism of Glc−11 formation.
Fig. 9 Putative activities of NicGS (BBL). Possible mechanistic routes towards (S)−1 from 10. The blue background shows key NaGR and NicGH reactions and major products. The grey background shows possible reactions that are either non-enzymatically or catalysed by NicGS. Only compounds with blue or white background have been directly detected. To induce the stereoselective outcome, NicGS may operate through: dynamic kinetic resolution (catalysing just purple arrows and allowing other intermediates to non-enzymatically equilibrate), epimerisation of 5b via a ring opened intermediate (catalysing red and purple arrows), catalysis of the stereoselective Mannich reaction between 3 and 10 prior to oxidation, reversal of the non-desired Mannich reaction (opening (R)-5b into 3 and 10). Blue arrows show the predicted mechanism of Glc−11 formation.CC BY · PubMed Central

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  • Highly accurate protein structure prediction with AlphaFold

    Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., Žídek, A., Potapenko, A., et al. (2021) · Nature · doi:10.1038/s41586-021-03819-2 · Cited by 43,764 · cited 1×

    “The structures were solved by molecular replacement using MOLREP 68 with the AlphaFold structures AF-P52579-F1-v4andAF-F1T160-F1-v4usedasthemodelsforNaGR and NicGS, respectively (https://alphafold.ebi.ac.uk/) 69 .”

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Metadata

Authors
Schwabe, B. T. W., Angstman, I. M., Vollheyde, K., Ingold, Z., Li, J., Stankevich, K. S., Spicer, C. D., Fascione, M. A., Grogan, G., Geu-Flores, F., & Lichman, B. R.
Affiliations
  • University of York (GB)
  • University of Copenhagen (DK)
Corresponding author
Lichman, Benjamin R.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2026-05-18
Volume
17
Issue
1
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-026-72705-0
PMC
PMC13184244
OpenAlex
W7161541149
Cited by
12
References
1
Citations by year
2026: 12
FWCI
24.63
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • Research at the University of Copenhagen (University of Copenhagen) (publishedVersion, cc-by)
  • White Rose Research Online (University of Leeds, The University of Sheffield, University of York) (acceptedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Research Councils UK (MR/S01862X/1, MR/X010260/1)
  • RCUK | Biotechnology and Biological Sciences Research Council (BB/T007222/1)
  • Det Frie Forskningsråd (5281-00267B)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant biochemistry and biosynthesis
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant tissue culture and regeneration
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant Gene Expression Analysis
Keywords
nicotine biosynthesis, tobacco, alkaloid biosynthesis, nicotinic acid, C–C bond formation, pyridine alkaloids, X-ray crystal structure
MeSH terms
Alkaloids, Glucose, Glycosylation, Nicotine, Plant Proteins, Nicotiana, Crystallography, X-Ray

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