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The first step in the biosynthesis of cocaine in Erythroxylum coca: the characterization of arginine and ornithine decarboxylases

Abstract

Despite the long history of cocaine use among humans and its social and economic significance today, little information is available about the biochemical and molecular aspects of cocaine biosynthesis in coca (Erythroxylum coca) in comparison to what is known about the formation of other pharmacologically-important tropane alkaloids in species of the Solanaceae. In this work, we investigated the site of cocaine biosynthesis in E. coca and the nature of the first step. The two principal tropane alkaloids of E. coca, cocaine and cinnamoyl cocaine, were present in highest concentrations in buds and rolled leaves. These are also the organs in which the rate of alkaloid biosynthesis was the highest based on the incorporation of 13CO2. In contrast, tropane alkaloids in the Solanaceae are biosynthesized in the roots and translocated to the leaves. A collection of EST sequences from a cDNA library made from young E. coca leaves was employed to search for genes encoding the first step in tropane alkaloid biosynthesis. Full-length cDNA clones were identified encoding two candidate enzymes, ornithine decarboxylase (ODC) and arginine decarboxylase (ADC), and the enzymatic activities of the corresponding proteins confirmed by heterologous expression in E. coli and complementation of a yeast mutant. The transcript levels of both ODC and ADC genes were highest in buds and rolled leaves and lower in other organs. The levels of both ornithine and arginine themselves showed a similar pattern, so it was not possible to assign a preferential role in cocaine biosynthesis to one of these proteins.

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Abbreviations

qRT–PCR:

Quantitative reverse transcriptase–polymerase chain reaction

LC–MS:

Liquid chromatography–mass spectrometry

ESTs:

Expressed sequence tags

NMR:

Nuclear magnetic resonance

GC–MS:

Gas chromatography–mass spectrometry

HPLC–UV–DAD:

High performance liquid chromatography–ultraviolet–diode array detection

HEPES:

(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)

ESI:

Electrospray ionization

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Acknowledgments

We thank Dr. Michael A. Phillips, for his help with the E. coca cDNA library, Katrin Luck for technical assistance, Dr Christian Paetz for help with the 13CO2 experiment, Gregor Schmidt for his help with qPCR normalization and Jan Jirschitzka for helping with the editing of the manuscript. In addition, we thank Prof Herbert Tabor for kindly providing the S. cerevisie ∆ mutant. Lastly, we thank Dr. Tamara Krügel, Andreas Weber and the rest of the gardening staff of the MPI-ICE for their help in plant maintenance. This work was supported by the Max Planck Society and an Alexander von Humboldt Foundation postdoctoral fellowship to J.D.

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Correspondence to John C. D’Auria.

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Docimo, T., Reichelt, M., Schneider, B. et al. The first step in the biosynthesis of cocaine in Erythroxylum coca: the characterization of arginine and ornithine decarboxylases. Plant Mol Biol 78, 599–615 (2012). https://doi.org/10.1007/s11103-012-9886-1

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Keywords

  • Erythroxylum coca
  • Tropane alkaloids
  • Cocaine
  • Ornithine and arginine decarboxylases