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Characterization of aromatic aminotransferases from Ephedra sinica Stapf

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Abstract

Ephedra sinica Stapf (Ephedraceae) is a broom-like shrub cultivated in arid regions of China, Korea and Japan. This plant accumulates large amounts of the ephedrine alkaloids in its aerial tissues. These analogs of amphetamine mimic the actions of adrenaline and stimulate the sympathetic nervous system. While much is known about their pharmacological properties, the mechanisms by which they are synthesized remain largely unknown. A functional genomics platform was established to investigate their biosynthesis. Candidate enzymes were obtained from an expressed sequence tag collection based on similarity to characterized enzymes with similar functions. Two aromatic aminotransferases, EsAroAT1 and EsAroAT2, were characterized. The results of quantitative reverse transcription-polymerase chain reaction indicated that both genes are expressed in young stem tissue, where ephedrine alkaloids are synthesized, and in mature stem tissue. Nickel affinity-purified recombinant EsAroAT1 exhibited higher catalytic activity and was more homogeneous than EsAroAT2 as determined by size-exclusion chromatography. EsAroAT1 was highly active as a tyrosine aminotransferase with α-ketoglutarate followed by α-ketomethylthiobutyrate and very low activity with phenylpyruvate. In the reverse direction, catalytic efficiency was similar for the formation of all three aromatic amino acids using l-glutamate. Neither enzyme accepted putative intermediates in the ephedrine alkaloid biosynthetic pathway, S-phenylacetylcarbinol or 1-phenylpropane-1,2-dione, as substrates.

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Acknowledgments

This research was supported by Research Grant No. CA-9117-09 from the Canada-Israel Binational Agricultural Research and Development Fund, the Natural Sciences and Engineering Research Council and Agriculture and Agri-Food Canada to E.L., P.F. and F.M. Granting agencies were not involved in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. K.K. was co-supervised by Norman Hüner during his M. Sc. program (Department of Biology, University of Western Ontario). We thank staff at the London Development and Research Centre, Agriculture and Agri-Food Canada, Tim McDowell for assistance with GC–MS, Sudhakar Pandurangan with RT-quantitative PCR, and Alex Molnar for the preparation of figures. We acknowledge support from the Genome Canada PhytoMetaSyn project for bioinformatic analysis.

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Authors and Affiliations

  1. Department of Biology, University of Western Ontario, London, ON, N6A 5B7, Canada

    Korey Kilpatrick & Frédéric Marsolais

  2. Agriculture and Agri-Food Canada, Genomics and Biotechnology, London Research and Development Centre, 1391 Sandford St., London, ON, N5V 4T3, Canada

    Korey Kilpatrick, Agnieszka Pajak, Mark W. Sumarah & Frédéric Marsolais

  3. Department of Biological Sciences, University of Calgary, Calgary, AB, Canada

    Jillian M. Hagel & Peter J. Facchini

  4. Department of Aromatic, Medicinal and Spice Crops, Newe Ya’ar Research Center, Agricultural Research Organization, 30095, Ramat Yishay, Israel

    Efraim Lewinsohn

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  1. Korey Kilpatrick
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  2. Agnieszka Pajak
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  3. Jillian M. Hagel
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Correspondence to Frédéric Marsolais.

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Kilpatrick, K., Pajak, A., Hagel, J.M. et al. Characterization of aromatic aminotransferases from Ephedra sinica Stapf. Amino Acids 48, 1209–1220 (2016). https://doi.org/10.1007/s00726-015-2156-1

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