Rat CYP2D2, not 2D1, is functionally conserved with human CYP2D6 in endogenous morphine formation

► Activity of rat CYP2D1 and 2D2 with three morphine precursors was tested. ► Thebaine and codeine were 3-O-demethylated by CYP2D2, not 2D1. ► CYP2D2 generated four phenol-coupled products from (R)-reticuline. ► CYP2D1 was completely inactive with (R)-reticuline. ► CYP2D2 showed high catalytic efficiencies for the three human CYP2D6 substrates. The assumption that CYP2D1 is the corresponding rat cytochrome to human CYP2D6 has been revisited using recombinant proteins in direct enzyme assays. CYP2D1 and 2D2 were incubated with known CYP2D6 substrates, the three morphine precursors thebaine, codeine and (R)-reticuline. Mass spectrometric analysis showed that rat CYP2D2, not 2D1, catalyzed the 3-O-demethylation reaction of thebaine and codeine. In addition, CYP2D2 incubated with (R)-reticuline generated four products corytuberine, pallidine, salutaridine and isoboldine while rat CYP2D1 was completely inactive. This intramolecular phenol-coupling reaction follows the same mechanism as observed for CYP2D6. Michaelis–Menten kinetic parameters revealed high catalytic efficiencies for rat CYP2D2. These findings suggest a critical evaluation of other commonly accepted, however untested, CYP2D1 substrates.