Characterization of recombinant betalain biosynthetic L-DOPA dioxygenase 1 (DOD1) from Basella alba L. 'Rubra' and classification of its paralogs.

Betalamic acid is part of the chromophore of betalains, which are pigments of chemotaxonomic and physiological importance. This study characterizes B. alba L. var. 'Rubra' L-DOPA/dopamine-4,5-dioxygenase 1 (BrDOD1). The results suggest that BrDOD1 is a unique high betalamic acid-forming enzyme among all reported LigB homologs in plants, with comparable affinity for both L-DOPA and dopamine (KM < 50 μM). Ascorbic acid (10 mM) increased both KM and Vmax by more than 6.5-fold, indicating that ascorbic acid acted possibly as an activity enhancer through local crowding, in addition to its role as a reducing agent. BrDOD1's physiological substrate is likely L-DOPA, as the reaction rate against L-DOPA was 6.6-fold higher than dopamine, L-DOPA was present in higher concentration than that of dopamine in the plant, and molecular dynamic simulations showed better stability of the complex of BrDOD1 with L-DOPA than that of dopamine. Two paralogs of BrDOD1 have also been cloned. Based on the betalamic acid-forming activity, molecular phylogeny, conserved structural regions, and theoretical pI, a classification of all betalainic plant LigB homologs into three groups has been proposed to better understand the physicochemical characteristics, structure-enzyme activity relationships, physiological roles, and evolutionary trajectory of plant LigB homologs. This work will contribute to the ongoing efforts of biotechnological production of betalains using BrDOD1 because of its low KM among reported DOD1s. BrDOD1 could be a potential component in biosensors for improved efficiency in detection of L-DOPA and/or dopamine.