Figure 5
From: Elucidation of the core betalain biosynthesis pathway in Amaranthus tricolor
Examination of the L-DOPA 4,5-dioxygenase activity of AmDODAα1 and AmDODAα2 in vitro. (a) Crude extracts prepared from E. coli expressing SUMO-fused BvDODAα1, AmDODAα1, or AmDODAα2 were examined by Coomassie brilliant blue staining. Recombinant proteins were expressed in similar amounts. (b) Enzymatic reactions were conducted following the method described by Sasaki et al32 with some modifications. The reaction mixtures contained L-DOPA, ascorbic acid, and the crude extract prepared from E. coli harboring the BvDODAα1, AmDODAα1, or AmDODAα2 gene. Yellow color derived from betalamic acid was examined to evaluate the enzymatic activity of L-DOPA 4,5-dioxygenase. (c) Elution profiles of the in vitro reactions were performed by LC–MS/MS. Betalamic acid was confirmed by a mass fragmentation profile of the peak at a retention time of 7.5 min.
