Abstract
Sanguinarine reductase is a plant enzyme that prevents the cytotoxic effects of benzophenanthridine alkaloids, which are the main phytoalexins of Papaveraceae. The enzyme catalyzes the reduction of sanguinarine, the most toxic benzophenanthridine, which re-enters the cytoplasm after its primary accumulation in the cell wall region has reached a threshold concentration. We present the sequence of the gene and protein of sanguinarine reductase isolated from cell cultures of Eschscholzia californica. High sequence similarities indicate that the enzyme evolved from a plant-specific branch of the ubiquitous Rossmann fold NAD(P)H/NAD(P)(+) binding reductases, with NADP-dependent epimerases or hydroxysteroid reductases as the most likely ancestors. Based on the x-ray structure of a close homolog, a three-dimensional model of the spatial conformation and catalytic site of sanguinarine reductase was established and used for in silico screening of known three-dimensional structures. Surprisingly, the enzyme shares high structural similarity with enzymes of human and bacterial origin, which have similar functions as the plant homologs but bear little amino acid sequence similarity. Using site-directed mutagenesis, a series of recombinant enzymes was generated and assayed to reveal the impact of individual amino acids and peptides in the catalytic process. It appears that relatively few innovations were required to generate this selective catalyst for alkaloid detoxication, notably an insertion of 13 amino acids and the generation of a novel catalytic triad of Cys-Asp-His were sufficient.