Characterization of two methylenedioxy bridge-forming cytochrome P450-dependent enzymes of alkaloid formation in the Mexican prickly poppy Argemone mexicana
Characterization of two methylenedioxy bridge-forming cytochrome P450-dependent enzymes of alkaloid formation in the Mexican prickly poppy Argemone mexicana
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DOI：
10.1016/j.abb.2010.11.016
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发表时间：
2011-03-01
影响因子：
3.9
通讯作者：
Kutchan, Toni M.
中科院分区：
文献类型：
--
作者：
Chavez, Maria Luisa Diaz;Rolf, Megan;Kutchan, Toni M.
亚甲二氧基桥的形成是罂粟科植物中苯并[c]菲啶和原小檗碱生物碱生物合成中不可或缺的步骤。植物中的这种反应由细胞色素P450依赖性酶催化。编码属于CYP 719家族的细胞色素P450酶的两个cDNA在询问从积累苯并[c]菲啶生物碱血根碱和原小檗碱生物碱小檗碱的墨西哥刺罂粟Argemone mexicana的2个月大的小植株制备的EST数据集后被鉴定。CYP 719 A13和CYP 719 A14与加州草木樨中苯并[c]菲啶生物合成的刺罂粟碱合酶CYP 719 A2的同源性分别为58%和77%和60%。CYP 719 A14和CYP 719 A13在草地贪夜蛾Sf 9细胞中的功能性异源表达产生了重组酶，其分别催化从（S）-石蒜碱形成（S）-cheilanthifoline的亚甲基二氧基桥和从（S）-cheilanthifoline形成（S）-stylopine的亚甲基二氧基桥。用每种酶测试27种潜在底物。而CYP 719 A14仅将（S）-scoulterine转化为（S）-cheilanthifoline（K-m 1.9 +/- 0.3; k（cat）/K-m 1.7），CYP 719 A13将（S）-四氢哥伦布胺转化为（S）-坎那丁（K-m 2.7 +/- 1.3; k（cat）/K-m，12.8），（S）-cheilanthifoline至（S）-stylopine（K-m 5.2 +/- 3.0; k（cat）/K-m 2.6）和（S）-苦味酸转化为（S）-南地宁（K-m 8.1 +/- 1.9; k（cat）/K-m 0.7）。这些结果表明，虽然CYP 719 A14只参与血根碱的生物合成，但CYP 719 A13可以参与血根碱和小檗碱的形成。墨西哥。(C)2010年爱思唯尔公司All rights reserved.
Formation of the methylenedioxy bridge is an integral step in the biosynthesis of benzo[c]phenanthridine and protoberberine alkaloids in the Papaveraceae family of plants. This reaction in plants is catalyzed by cytochrome P450-dependent enzymes. Two cDNAs that encode cytochrome P450 enzymes belonging to the CYP719 family were identified upon interrogation of an EST dataset prepared from 2-month-old plantlets of the Mexican prickly poppy Argemone mexicana that accumulated the benzo[c]phenanthridine alkaloid sanguinarine and the protoberberine alkaloid berberine. CYP719A13 and CYP719A14 are 58% identical to each other and 77% and 60% identical, respectively, to stylopine synthase CYP719A2 of benzo[c]phenanthridine biosynthesis in Eschscholzia californica. Functional heterologous expression of CYP719A14 and CYP719A13 in Spodoptera frugiperda Sf9 cells produced recombinant enzymes that catalyzed the formation of the methylenedioxy bridge of (S)-cheilanthifoline from (S)-scoulerine and of (S)-stylopine from (S)-cheilanthifoline, respectively. Twenty-seven potential substrates were tested with each enzyme. Whereas CYP719A14 transformed only (S)-scoulerine to (S)-cheilanthifoline (K-m 1.9 +/- 0.3; k(cat)/K-m 1.7), CYP719A13 converted (S)-tetrahydrocolumbamine to (S)-canadine (K-m 2.7 +/- 1.3; k(cat)/K-m, 12.8), (S)-cheilanthifoline to (S)-stylopine (K-m 5.2 +/- 3.0; k(cat)/K-m 2.6) and (S)-scoulerine to (S)-nandinine (K-m 8.1 +/- 1.9; k(cat)/K-m 0.7). These results indicate that although CYP719A14 participates in only sanguinarine biosynthesis, CYP719A13 can be involved in both sanguinarine and berberine formation in A. mexicana. (C) 2010 Elsevier Inc. All rights reserved.