A cyclo‐ DOPA 6‐ O ‐glucosyltransferase‐mediated route for gomphrenin I biosynthesis in Basella alba and Gomphrena globosa

Betacyanins are red pigments characteristic of Caryophyllales and show considerable structural diversity, yet the enzymatic basis underlying 6-O-glucosylated betacyanins, such as gomphrenin I, has remained unclear. In particular, how alternative glucosylation patterns contribute to betacyanin diversification is poorly understood. Here, we identified cyclo-DOPA glucosyltransferases from Basella alba and Gomphrena globosa and examined their roles in gomphrenin I biosynthesis using transient expression assays and tobacco BY-2 cell systems. Phylogenetic analyses, structural modelling, and site-directed mutagenesis were employed to investigate their functional and structural characteristics. BacDOPA5/6GTs catalysed both 5-O- and 6-O-glucosylation of cyclo-DOPA, leading to the production of betanin and gomphrenin I, whereas GgcDOPA6GT specifically mediated gomphrenin I formation. These enzymes belong to distinct subclades within the cDOPA-GT family, and mutational analyses demonstrated essential roles for conserved histidine residues and an α-helical region adjacent to the catalytic site. Thermal stability analyses further showed that gomphrenin I is more thermally stable than betanin, likely due to the formation of an intramolecular hydrogen bond. Together, these results reveal an additional cDOPA6GT-mediated route for gomphrenin I biosynthesis and provide insight into the diversification and functional specialisation of betacyanins, linking the position of glucosylation to pigment stability and biochemical properties.