Structure and Mechanism of Sanguinarine Reductase, an Enzyme of Alkaloid Detoxification

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.

Interaction of the Anticancer Plant Alkaloid Sanguinarine with Bovine Serum Albumin

“…This may involve strong H-bonding by the OH at the C6 position of SA to amino acids of BSA. Such an H-bonding has been shown to occur in sanguinarine-reductase-SA interactions [62]. A detailed analysis of the various contributions to Δ C p is beyond the scope of this paper and is not attempted.…”

Short‐chain dehydrogenase/reductase catalyzing the final step of noscapine biosynthesis is localized to laticifers in opium poppy

“…In addition to NOS, two other SDR-type enzymes have been implicated in BIA metabolism: SanR (Vogel et al, 2010) and SalR (Ziegler et al, 2006). SanR catalyzes the NAD(P)H-dependent reduction of sanguinarine to dihydrosanguinarine (Vogel et al, 2010). Although SanR is capable of using either NADH or NADPH as a hydrogen donor, the catalytic rate with NADPH is substantially higher than with NADH (Vogel et al, 2010).…”

Morphine Biosynthesis in Opium Poppy Involves Two Cell Types: Sieve Elements and Laticifers

“…Only the epimerization of reticuline has not been characterized at the molecular biochemical level (De-Eknamkul and Zenk, 1992). Additional cDNAs encoding several other BIA biosynthetic enzymes have been characterized from opium poppy (see Supplemental Figure 1 online), including reticuline 7-O-methyltransferase (7OMT) (Ounaroon et al, 2003), norreticuline 7-O-methyltransferase (N7OMT) (Pienkny et al, 2009), scoulerine O-methyltransferase (SOMT) (Dang and Facchini, 2012;Winzer et al, 2012), berberine bridge enzyme (Facchini et al, 1996), stylopine synthase (Díaz-Chávez et al, 2011), tetrahydroprotoberberine N-methyltransferase (TNMT) (Liscombe and Facchini, 2007), pavine N-methyltransferase (PavNMT) (Liscombe et al, 2009), and sanguinarine reductase (Vogel et al, 2010).…”