In vitro functional reconstitution of cofactor-dependent plant cytochrome P450 system on artificial liposomes

Cytochrome P450s (CYPs) and their associated reductases (CPRs) play a central role in plant secondary metabolism. These enzymes operate on lipid bilayers and require cofactors such as heme, FAD, and FMN. In this study, we developed an in vitro system to reconstitute CYPs and CPRs as functional enzymes on liposomes. As model proteins, we selected cinnamate 4-hydroxylase (C4H) from Arabidopsis thaliana and its redox partner ATR1. We investigated optimal conditions for incorporating cofactors into C4H and ATR1 and successfully reconstituted their catalytic activity. Hematin was found to be more effective than hemin as a heme source for C4H activation. Both C4H and ATR1 were embedded in liposomes, and the catalytic activity of C4H was significantly enhanced when both proteins were co-synthesized. These results highlight the importance of functional protein–protein interaction between C4H and ATR1 in achieving efficient electron transfer and catalytic function in vitro.