title: Characterization of aromatic aminotransferases from Ephedra sinica Stapf.
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.
authors.affiliation: Department of Biology, University of Western Ontario, London, ON, N6A 5B7, Canada.|Agriculture and Agri-Food Canada, Genomics and Biotechnology, London Research and Development Centre, 1391 Sandford St., London, ON, N5V 4T3, Canada.
authors.forename: Korey
authors.initials: K
authors.lastname: Kilpatrick
authors.affiliation: Agriculture and Agri-Food Canada, Genomics and Biotechnology, London Research and Development Centre, 1391 Sandford St., London, ON, N5V 4T3, Canada.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Pajak
authors.affiliation: Department of Biological Sciences, University of Calgary, Calgary, AB, Canada.
authors.forename: Jillian M
authors.initials: JM
authors.lastname: Hagel
authors.affiliation: Agriculture and Agri-Food Canada, Genomics and Biotechnology, London Research and Development Centre, 1391 Sandford St., London, ON, N5V 4T3, Canada.
authors.forename: Mark W
authors.initials: MW
authors.lastname: Sumarah
authors.affiliation: Department of Aromatic, Medicinal and Spice Crops, Newe Ya'ar Research Center, Agricultural Research Organization, 30095, Ramat Yishay, Israel.
authors.forename: Efraim
authors.initials: E
authors.lastname: Lewinsohn
authors.affiliation: Department of Biological Sciences, University of Calgary, Calgary, AB, Canada.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Facchini
authors.affiliation: Department of Biology, University of Western Ontario, London, ON, N6A 5B7, Canada. Frederic.Marsolais@agr.gc.ca.|Agriculture and Agri-Food Canada, Genomics and Biotechnology, London Research and Development Centre, 1391 Sandford St., London, ON, N5V 4T3, Canada. Frederic.Marsolais@agr.gc.ca.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Marsolais
mesh_terms: D055162:Biocatalysis; D004795:Enzyme Stability; D029790:Ephedra sinica / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004809:Ephedrine / Q000378:metabolism; D007700:Kinetics; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000637:Transaminases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D000637:Transaminases; D004809:Ephedrine
keywords: Aminotransferase; Aromatic amino acids; Ephedra sinensis Stapf Ephedraceae; L-Tyrosine; Pyridoxal 5′-phosphate; Transcriptome analysis