Detection of a novel intramolecular rearrangement during gramine biosynthesis in barley using stable isotope-labeled tryptophan

Plants accumulate various secondary metabolites, and the biosynthetic reactions responsible for their scaffold construction are the key steps that characterize their structural categories. Gramine, an indole alkaloid, is a defensive secondary metabolite biosynthesized in barley (Hordeum vulgare) from tryptophan (Trp) via aminomethylindole (AMI). While the two sequential N-methylation steps following the formation of AMI have already been characterized both genetically and enzymatically, the step preceding AMI formation, which includes the Trp side chain-shortening, has not yet been revealed. To gain further insight into these biosynthetic reactions, barley seedlings were fed Trp labeled with stable isotopes ((13)C and (15)N) at various positions, and the isotope incorporation into gramine was analyzed by liquid chromatography/mass spectrometry. Significant increases in the abundance of isotopic gramine were detected in experimental sets in which Trp was labeled at either the indole ring, the β-carbon, or the amino group, whereas the isotopolog composition was not affected by α-carbon-labeled Trp. Although absorbed Trp presumably undergoes transamination in plants, this reaction did not seem to be related to gramine productivity. The data indicated that AMI directly inherited the amino group from Trp, while the α-carbon was removed, suggesting that the Trp–AMI conversion includes a novel intramolecular rearrangement reaction. The results of this study provide novel insights into scaffold formation in plant secondary-metabolite synthesis.