Abstract
Many specialized metabolic pathways have evolved convergently in plants, but distinguishing multiple origins from alternative evolutionary scenarios can be difficult. Here, we explore the evolution of l-3,4-dihydroxyphenylalanine (l-DOPA) 4,5-dioxygenase (DODA) enzymes to better resolve the convergent evolution of the betalain biosynthetic pathway within the flowering plant order Caryophyllales. We use yeast-based heterologous assays to quantify enzymatic activity of extant proteins and then employ ancestral sequence reconstruction to resurrect and assay ancestral DODA enzymes. We use a combination of ancestral sequence reconstruction, model-based methods, and structural modelling to describe patterns of molecular convergence. We confirm that high l-DOPA 4,5-dioxygenase activity is polyphyletic and show that high activity DODAs evolved at least three times from ancestral proteins with low activity. We show that molecular convergence is concentrated proximally to the binding pockets but also appears distally to active sites. Moreover, our analysis also suggests that many unique and divergent substitutions contribute to the evolution of DODA. Given the key role of DODA in betalain biosynthesis, our analysis further supports the convergent origins of betalains and illustrates how the iterative evolution of betalain biosynthesis has drawn on a complex mixture of convergent, divergent, and unique variation.