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Heterologous expression of two FAD-dependent oxidases with (S)-tetrahydroprotoberberine oxidase activity from Arge mone mexicana and Berberis wilsoniae in insect cells

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Abstract

Berberine, palmatine and dehydrocoreximine are end products of protoberberine biosynthesis. These quaternary protoberberines are elicitor inducible and, like other phytoalexins, are highly oxidized. The oxidative potential of these compounds is derived from a diverse array of biosynthetic steps involving hydroxylation, intra-molecular C–C coupling, methylenedioxy bridge formation and a dehydrogenation reaction as the final step in the biosynthesis. For the berberine biosynthetic pathway, the identification of the dehydrogenase gene is the last remaining uncharacterized step in the elucidation of the biosynthesis at the gene level. An enzyme able to catalyze these reactions, (S)-tetrahydroprotoberberine oxidase (STOX, EC 1.3.3.8), was originally purified in the 1980s from suspension cells of Berberis wilsoniae and identified as a flavoprotein (Amann et al. 1984). We report enzymatic activity from recombinant STOX expressed in Spodoptera frugiperda Sf9 insect cells. The coding sequence was derived successively from peptide sequences of purified STOX protein. Furthermore, a recombinant oxidase with protoberberine dehydrogenase activity was obtained from a cDNA library of Argemone mexicana, a traditional medicinal plant that contains protoberberine alkaloids. The relationship of the two enzymes is discussed regarding their enzymatic activity, phylogeny and the alkaloid occurrence in the plants. Potential substrate binding and STOX-specific amino acid residues were identified based on sequence analysis and homology modeling.

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Notes

  1. Molecular mass of coreximine used in this study is m/z 330, since only isotopic labeled coreximine was available. The label(s) are part of the isoquinoline moiety.

Abbreviations

CDS:

Coding sequence

STOX:

(S)-Tetrahydroprotoberberine oxidase

BBE:

Berberine bridge enzyme

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Acknowledgments

The authors acknowledge Christian Böttcher (Biozentrum, Halle (Saale), GER) for technical assistance in the purification of STOX protein. We thank Prof. Dr. Jürgen Ehlting (UVIC, Victoria BC, Canada) for interesting discussions on phylogenetics and Russ Chedgy (do.) for critical reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn, Fonds der Chemischen Industrie, Frankfurt, the German Academic Exchange Service (DAAD), Bonn and Consejo Nacional de Ciencia y Tecnología (CONACyT) and Programa de Mejoramiento del Profesorado/Universidad Autónoma del Estado de Morelos (PROMEP/UAEM), Mexico.

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Authors and Affiliations

  1. Leibniz-Institut für Pflanzenbiochemie, Halle (Saale), Germany

    Andreas Gesell, Maria Luisa Díaz Chávez, Robert Kramell & Toni M. Kutchan

  2. Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, MO, 63132, USA

    Maria Luisa Díaz Chávez & Toni M. Kutchan

  3. Department of Plant Physiology, Ruhr-Universität Bochum, Bochum, Germany

    Markus Piotrowski

  4. Institute of Biochemistry, Graz University of Technology, Graz, Austria

    Peter Macheroux

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  1. Andreas Gesell
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  2. Maria Luisa Díaz Chávez
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  3. Robert Kramell
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  4. Markus Piotrowski
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  5. Peter Macheroux
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  6. Toni M. Kutchan
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Correspondence to Toni M. Kutchan.

Additional information

A. Gesell and M. L. Díaz Chávez contributed equally to the paper.

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425_2011_1357_MOESM1_ESM.txt

Suppl. File S1 Translated Aquilegia coerulea contigs and Papaver somniferum Ps_cntg1 Supplementary material 1 (TXT 5 kb)

425_2011_1357_MOESM2_ESM.nwk

Suppl. File S2 Phylogenetic tree file of Fig. 6a. Abbreviations: NCBI GenBank numerical identifiers following of a letter code for taxonomy (NWK 4 kb)

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Gesell, A., Díaz Chávez, M.L., Kramell, R. et al. Heterologous expression of two FAD-dependent oxidases with (S)-tetrahydroprotoberberine oxidase activity from Arge mone mexicana and Berberis wilsoniae in insect cells. Planta 233, 1185–1197 (2011). https://doi.org/10.1007/s00425-011-1357-4

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