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

植物保护细胞免受自身产生的毒素毒害的能力是植物抗毒素进化成功的一个重要但较少被研究的方面。苯并菲并菲生物碱是一种细胞毒性抗菌剂，主要存在于罂粟科植物的根部。在培养的加州棘球藻细胞中，我们证明了这些生物碱分泌到质外体和外部培养基中。外部生物碱，尤其是高度细胞毒性的血根碱，经历了一个循环，包括快速的再吸收和相应的还原成毒性较小的二氢生物碱。血根碱还原酶是我们实验室发现和克隆的一种新的细胞质酶，它的摄取是由血根碱还原酶驱动的。与拟南芥和水稻相似的蛋白质相比，血根碱还原酶含有独特的序列基序，可能参与了高度特异的催化作用。这个项目计划完成1。通过基因沉默证实血根碱还原酶在培养细胞中的解毒作用。产生的Eschschizia菌株被用来测试还原酶和生物碱生物合成以及AS2的共同调节。揭示苯并二氮杂菲的跨膜转运机制，不依赖于它们的还原和进一步的代谢。通过三维结构分析、同源建模和定点突变，揭示催化所必需的酶蛋白的结构元件；4.在一系列植物物种中寻找血根素还原酶和苯并菲并菲生物合成共同进化的线索。

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.