Hordeum vulgare CYP76M57 catalyzes C2 shortening of tryptophan side chain by C–N bond rearrangement in gramine biosynthesis

SUMMARY The indole alkaloid gramine, 3‐(dimethylaminomethyl)indole, is a defensive specialized metabolite found in some barley cultivars. In its biosynthetic process, the tryptophan (Trp) side chain is shortened by two carbon atoms to produce 3‐(aminomethyl)indole (AMI), which is then methylated by N ‐methyltransferase (HvNMT) to produce gramine. Although side chain shortening is one of the crucial scaffold formation steps of alkaloids originating from aromatic amino acids, the gene and enzyme involved in the Trp–AMI conversion reactions are unknown. In this study, through RNA‐seq analysis, 35 transcripts were shown to correlate with gramine production; among them, an uncharacterized cytochrome P450 (CYP) gene, CYP76M57 , and HvNMT were identified as candidate genes for gramine production. Transgenic Arabidopsis thaliana and rice overexpressing CYP and HvNMT accumulate AMI, N ‐methyl‐AMI, and gramine. CYP76M57, heterologously expressed in Pichia pastoris , was able to act on Trp to produce AMI. Furthermore, the amino group nitrogen of Trp was retained during the CYP76M57‐catalyzed reaction, indicating that the C 2 shortening of Trp proceeds with an unprecedented biosynthetic process, the removal of the carboxyl group and C α and the rearrangement of the nitrogen atom to C β . In some gramine‐non‐accumulating barley cultivars, arginine 104 in CYP76M57 is replaced by threonine, which abolished the catalytic activity of CYP76M57 to convert Trp into AMI. These results uncovered the missing committed enzyme of gramine biosynthesis in barley and contribute to the elucidation of the potential functions of CYPs in plants and undiscovered specialized pathways.