Abstract
The two types of red pigment, anthocyanins and betacyanins, never coexist in the same plant. While the anthocyanin biosynthetic genes have been cloned and analyzed, the betacyanin biosynthetic pathway is still poorly understood. We carried out the biochemical analysis of two Phytolacca americana DOPA dioxygenases (PaDOD1 and PaDOD2) that may be involved in betalain biosynthesis. The recombinant protein of PaDOD1 catalyzed the conversion of DOPA to betalamic acid, whereas DOD activity was not detected in PaDOD2 in vitro. While the reported motif conserved in DODs from betalain-producing plants was found in PaDOD1, a single amino acid residue alteration was detected in PaDOD2. The site-directed mutagenesis experiments suggested the contribution of a conserved motif to full DOD activity and the presence of another essential element(s) for dioxygenase activity. The chimeric proteins of PaDOD1 and PaDOD2 were constructed and analyzed to identify the essential regions for DOD activity. Multiple essential regions for DOPA dioxygenase activity were found in PaDOD1. The evolutionary mechanism of DOD genes is discussed using phylogenetic analysis.
