Cloning and Functional Characterization of NADPH-Cytochrome P450 Reductases in .

Diterpenoid alkaloids (DAs) are major pharmacologically active ingredients of , an important medicinal plant. Cytochrome P450 monooxygenases (P450s) are involved in the DA biosynthetic pathway, and the electron transfer reaction of NADPH-cytochrome P450 reductase (CPR) with P450 is the rate-limiting step of the P450 redox reaction. Here, we identified and characterized two homologs of CPR from . The open reading frames of and were found to be 2103 and 2100 bp, encoding 700 and 699 amino acid residues, respectively. Phylogenetic analysis characterized both and as class II CPRs. Cytochrome and ferricyanide could be reduced with the recombinant proteins of AvCPR1 and AvCPR2. Both and were expressed in the roots, stems, leaves, and flowers of . The expression levels of and were significantly increased in response to methyl jasmonate (MeJA) treatment. The yeasts co-expressing // and all produced ferruginol, indicating that AvCPR1 and AvCPR2 can transfer electrons to CYP76AH1 in the same manner as SmCPR1. Docking analysis confirmed the experimentally deduced functional activities of and for FMN, FAD, and NADPH. The functional characterization of s will be helpful in disclosing molecular mechanisms relating to the biosynthesis of diterpene alkaloids in .