CS-6-1 - Codeinone reductase isoforms with differential stability, efficiency and product specificity in opium poppy
Sunday, July 15
3:18 PM - 3:38 PM
Opium poppy (Papaver somniferum) has been used as a medicinal plant since before the dawn of civilization; today it remains the major commercial source of natural opiate analgesics morphine and codeine, and semisynthetic derivatives, including naloxone. Microbial production of opioids would create a significant industrial advantage and provide an avenue for production of tailored opioids with reduced addictive properties. Codeinone reductase (COR) catalyzes the reversible NADPH-dependent reduction of codeinone to codeine in the penultimate step of morphine biosynthesis in opium poppy. The substrate, codeinone, exists in an equilibrium with its positional isomer, neopinone, in an aqueous solution. COR can alternatively reduce neopinone to neopine. In a parallel pathway involving 3-O-desmethylated analogs, COR also converts morphinone to morphine, and neomorphinone to neomorphine. Microbial platforms expressing COR lead to an overwhelming accumulation of the undesired by-products, neopine and neomorphine; in contrast, the plant only produces minimal levels of neopine. We have analyzed a range of COR isoforms from opium poppy varieties and isolated an isoform COR-B that shows superlative catalytic activity. We have discovered that all CORs favour the reduction of codeinone to codeine; however, the reduction of neopinone to neopine occurs irreversibly, leading to the gradual accumulation of this undesired alkaloid. Through site-directed mutagenesis we dissected the various biochemical characteristics of COR isoforms and discovered that greater catalytic proficiency, including protein stability, cofactor affinity and reaction velocity, are inextricably linked to the greater production of neopine. We identified four key residues that affected these characteristics. This knowledge provides great insights into sequence-function relationships of COR isoforms and can result in a ten-fold increase in metabolic output, when translated into engineered yeast.
Peter Facchini – University of Calgary