Molecular cloning of melatonin 2‐hydroxylase responsible for 2‐hydroxymelatonin production in rice ( O ryza sativa )

Abstract Although melatonin biosynthetic genes from plants have been cloned, the melatonin catabolism mechanisms remain unclear. To clone the genes responsible for melatonin metabolism, we ectopically expressed 35 full‐length cDNA s of rice 2‐oxoglutarate‐dependent dioxygenase (2‐ ODD ) in Escherichia coli and purified the corresponding recombinant proteins. In vitro 2‐ ODD assays showed four independent 2‐ ODD proteins that were able to catalyze melatonin into 2‐hydroxymelatonin, exhibiting melatonin 2‐hydroxylase (M2H). These M2H proteins had peak activities at pH 8.0 and 30°C. The K m ranged from 121  μ m to 371  μ m with the V max ranging from 1.7 to 18.5 pkat/mg protein, respectively. The M2H enzyme activities were dependent on cofactors such as α ‐ketoglutarate, ascorbate, and Fe 2+ , similar to the 2‐ ODD enzymes. M2H activity was inhibited by prohexadione‐Ca, an inhibitor of 2‐ ODD , in a dose‐dependent manner. M2H activity was high in the roots of rice seedlings, concurrent with high transcription levels of 2‐ ODD 21 , suggesting that 2‐ ODD 21 was a major gene for M2H activity. Analogous to the high M2H activity in the roots, 2‐hydroxymelatonin was found in large quantities in roots treated with melatonin. These results suggest that melatonin was metabolized into 2‐hydroxymelatonin by the M2H genes in plants, but the physiological significance of 2‐hydroxymelatonin remains to be examined in the future.