CLONING AND HETEROLOGOUS EXPRESSION OF NADPH-CYTOCHROME P450 REDUCTASES FROM THE PAPAVERACEAE

Cytochrome P450 reductase was purified to homogeneity from cell suspen sion cultures of the opium poppy Papaver somniferum, the enzyme was ch aracterized (K-m cytochrome c, 8.3 mu M; K-m NADPH, 4.2 mu M; PH optim um, 8.0; M-r, 80 kDa), and the amino acid sequence of internal peptide s was determined. Partial cDNA clones hom P. somniferum and from Eschs cholzia californica (California poppy) were then generated using the p olymerase chain reaction and were used as hybridization probes to isol ate full-length cDNAs. The Papaver and Eschscholzia cytochrome P450 re ductases are 63% identical at the nucleotide level and 69% identical a t the amino acid level. SDS-PAGE of the purified native P. somniferum enzyme as well as genomic DNA gel blot analysis indicate that two cyto chrome P450 reductase isoforms are present in each species. This evide nce is also supported by translation of nucleotide sequences obtained hom the PCR-generated partial cDNAs and the full-length cDNAs isolated from lambda libraries. The Papaver and Eschscholzia cyto chrome P450 reductases were functionally expressed in the yeast Saccharomyces cere visiae and in the insect cell culture Spodoptera frugiperda Sf9. Coexp ression of cytochrome P450 reductase with the C-O phenol coupling cyto chrome P450 of bisbenzylisoquinoline alkaloid biosynthesis in Berberis stolonifera, berbamunine synthase (CYP80A1), in insect cell culture r esulted in an alteration of the product profile as compared to that ob tained by expression of berbamunine synthase in the absence Of plant r eductase. (C) 1997 Academic Press.