Cloning and Heterologous Expression of NADPH-Cytochrome P450 Reductases from the Papaveraceae

Cytochrome P450 reductase was purified to homogeneity from cell suspension cultures of the opium poppyPapaver somniferum,the enzyme was characterized (Kmcytochromec,8.3 μM;KmNADPH, 4.2 μM; pH optimum, 8.0;Mr, 80 kDa), and the amino acid sequence of internal peptides was determined. Partial cDNA clones fromP. somniferumand fromEschscholzia californica(California poppy) were then generated using the polymerase chain reaction and were used as hybridization probes to isolate full-length cDNAs. ThePapaverandEschscholziacytochrome P450 reductases are 63% identical at the nucleotide level and 69% identical at the amino acid level. SDS–PAGE of the purified nativeP. somniferumenzyme as well as genomic DNA gel blot analysis indicate that two cytochrome P450 reductase isoforms are present in each species. This evidence is also supported by translation of nucleotide sequences obtained from the PCR-generated partial cDNAs and the full-length cDNAs isolated from λ libraries. ThePapaverandEschscholziacytochrome P450 reductases were functionally expressed in the yeastSaccharomyces cerevisiaeand in the insect cell cultureSpodoptera frugiperdaSf9. Coexpression of cytochrome P450 reductase with theC–Ophenol coupling cytochrome P450 of bisbenzylisoquinoline alkaloid biosynthesis inBerberis stolonifera,berbamunine synthase (CYP80A1), in insect cell culture resulted in an alteration of the product profile as compared to that obtained by expression of berbamunine synthase in the absence of plant reductase.