Sanguinarine reductase - molecular and cellular characterization of a key enzyme in the detoxification of benzophenanthridine

血根碱还原酶 - 苯并菲啶解毒关键酶的分子和细胞表征

• 批准号: 61027654
• 负责人: Professor Dr. Werner Roos
• 金额: --
• 依托单位: Abteilung Molekulare Zellbiologie (aufgelöst)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2008
• 资助国家: 德国
• 起止时间: 2007-12-31 至 2010-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/61027654?language=en
• 关键词: Sanguinarine; reductase; molecular; cellular; characterization

The ability of plants to protect their cells from toxic effects of self-produced toxins is an important, but less investigated aspect of the evolutionary success of phytoalexins. Benzophenanthridine alkaloids are cytotoxic antimicrobials that are mainly built in the roots of Papaveraceae. In cultured cells of Eschscholzia californica we have demonstrated the excretion of these alkaloids into the apoplast and outer medium. External alkaloids, preferentially the highly cytotoxic sanguinarine, undergo a recycling that includes rapid reuptake and concomitant reduction to less toxic dihydro-alkaloids. Uptake is driven by sanguinarine reductase, a novel, cytoplasmic enzyme discovered and cloned in our laboratory. Compared to similar proteins of Arabidopsis and Oryza, sanguinarine reductase contains unique sequence motifs, that are likely involved in the highly specific catalysis. The project intends1. to prove the putative detoxifying function of sanguinarine reductase by gene silencing in cultured cells. The resulting Eschschoizia strains are used to test for co-regulation of the reductase and the alkaloid biosynthesis as well as2. to reveal the transport mechanisms of benzophenanthhdines across the plasma membrane, independent of their reduction and further metabolization.3. to reveal the structural elements of the enzyme protein that are essential for catalysis, by 3D structural analysis, homology modeling and site-directed mutagenesis,4. to search for hints of a co-evolution of sanguinarin reductase and benzophenanthridine biosynthesis among a selection of plant species.

植物保护其细胞免受自身产生的毒素的毒性作用的能力是植物抗毒素进化成功的一个重要但较少研究的方面。苯并菲啶生物碱是细胞毒性抗菌剂，主要存在于罂粟科的根部。在加州大肠杆菌的培养细胞中，我们已经证明这些生物碱会分泌到质外体和外部培养基中。外来生物碱，特别是高细胞毒性的血根碱，会经历再循环，包括快速再摄取并同时还原为毒性较小的二氢生物碱。吸收是由血根碱还原酶驱动的，这是我们实验室发现并克隆的一种新型细胞质酶。与拟南芥和稻的类似蛋白相比，血根碱还原酶含有独特的序列基序，可能参与高度特异性的催化。该项目的目的1。通过培养细胞中的基因沉默来证明血根碱还原酶的假定解毒功能。所得 Eschschoizia 菌株用于测试还原酶和生物碱生物合成的共同调节以及2。揭示苯并菲啶跨质膜的转运机制，与其还原和进一步代谢无关。3．通过3D结构分析、同源建模和定点诱变揭示催化所必需的酶蛋白的结构元件，4。在选定的植物物种中寻找血根碱还原酶和苯并菲啶生物合成共同进化的线索。