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

• Applicant: Professor Dr. Werner Roos
• Subject Area: Plant Cell and Developmental Biology
• Term: from 2008 to 2011
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 61027654

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.

• DFG Programme: Research Grants