Basella alba L. var. Rubra L-DOPA/dopamine-4,5-dioxygenase 1 prefers L-DOPA over dopamine and ascorbic acid enhances its activity

Betalamic acid is the chromophore of betalains, which are pigments of chemotaxonomic and physiological importance. This study involves RACE-PCR-based gene cloning, heterologous expression, protein purification, and steady-state kinetics of B. alba L. var. Rubra L-DOPA/dopamine-4,5-dioxygenase 1 (BrDOD1). BrDOD1 is a unique high betalamic acid-forming LigB homolog in plants having comparable affinity for both L-DOPA and dopamine (KM < 50 M). Ascorbic acid (10 mM) shifted the steady-state kinetics from inhibitory to activator at a particular substrate concentration of both L-DOPA and dopamine. This increased both KM and Vmax by more than 6.5-fold, indicating that ascorbic acid acted as a molecular crowding agent in the enzyme assay. BrDOD1s physiological substrate is L-DOPA as the reaction rate for L-DOPA was 6.6-fold higher than dopamine, L-DOPA was present in higher concentration than that of dopamine in the same plant, and molecular dynamic simulations showed better stability of BrDOD1-L-DOPA complex than that of dopamine. Further, two more LigB homologs from the same plant have also been cloned. Based on the betalamic acid-forming activity, molecular phylogeny, conserved structural regions, and theoretical pI, betalainic plant LigB homologs have been classified into three groups to better understand the evolutionary trajectory of the LigB homologs in plants.