Characterization of aromatic aminotransferases from Ephedra sinica Stapf
Characterization of aromatic aminotransferases from Ephedra sinica Stapf
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DOI：
10.1007/s00726-015-2156-1
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发表时间：
2016-05-01
期刊：
影响因子：
3.5
通讯作者：
Marsolais, Frederic
中科院分区：
文献类型：
--
作者：
Kilpatrick, Korey;Pajak, Agnieszka;Marsolais, Frederic
草麻黄（Ephedra sinica Stapf）是麻黄科（Ephedraceae）植物麻黄属（Ephedra sinica）的一种帚状灌木，广泛分布于中国、朝鲜和日本的干旱地区。这种植物在其气生组织中积累了大量的麻黄碱生物碱。这些安非他明类似物模仿肾上腺素的作用，刺激交感神经系统。虽然对它们的药理学特性有很多了解，但它们的合成机制在很大程度上仍然未知。建立了一个功能基因组学平台，以研究其生物合成。基于与具有相似功能的表征酶的相似性，从表达的序列标签集合获得候选酶。两个芳香族氨基转移酶，EsAroAT 1和EsAroAT 2的特点。定量逆转录-聚合酶链反应的结果表明，这两个基因表达在年轻的茎组织，麻黄碱生物碱的合成，并在成熟的茎组织。镍亲和纯化的重组EsAroAT 1表现出更高的催化活性，更均匀比EsAroAT 2的尺寸排阻色谱法测定。EsAroAT 1作为酪氨酸氨基转移酶与α-酮戊二酸、随后与α-酮甲基硫代丁酸具有高度活性，与苯丙酮酸具有非常低的活性。在相反的方向上，催化效率是相似的形成所有三个芳香族氨基酸使用L-谷氨酸。两种酶都不接受麻黄碱生物碱生物合成途径中的假定中间体，S-苯乙酰基甲醇或1-苯基丙烷-1，2-二酮，作为底物。
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 alpha-ketoglutarate followed by alpha-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.