Heterologous expression of two FAD-dependent oxidases with (S)-tetrahydroprotoberberine oxidase activity from Arge mone mexicana and Berberis wilsoniae in insect cells
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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DOI：
10.1007/s00425-011-1357-4
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发表时间：
2011-06-01
期刊：
影响因子：
4.3
通讯作者：
Kutchan, Toni M.
中科院分区：
文献类型：
--
作者：
Gesell, Andreas;Diaz Chavez, Maria Luisa;Kutchan, Toni M.
小檗碱、巴马汀和脱氢coreximine是原小檗碱生物合成的终产物。这些四级原小檗碱是激发子诱导的，并且像其他植物抗毒素一样，是高度氧化的。这些化合物的氧化电位来源于一系列不同的生物合成步骤，包括羟基化、分子内C-C偶联、亚甲二氧基桥形成和作为生物合成最后步骤的脱氢反应。对于小檗碱生物合成途径，脱氢酶基因的鉴定是在基因水平上阐明生物合成的最后剩余的未表征的步骤。一种能够催化这些反应的酶，（S）-四氢原小檗碱氧化酶（STOX，EC 1.3.3.8），最初在20世纪80年代从小黄连素的悬浮细胞中纯化，并被鉴定为黄素蛋白（Amann等，1984）。我们报道了在草地贪夜蛾Sf 9昆虫细胞中表达的重组STOX的酶活性。编码序列依次衍生自纯化的STOX蛋白的肽序列。此外，从含有原小檗碱生物碱的传统药用植物-墨西哥银仔（Argemone mexicana）的cDNA文库中获得了具有原小檗碱脱氢酶活性的重组氧化酶。从酶的活性、生物碱的发生和植物中生物碱的存在等方面讨论了这两种酶的关系。潜在的底物结合和STOX特异性氨基酸残基的基础上，序列分析和同源性建模进行了鉴定。
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.