IDENTIFICATION AND CHARACTERIZATION OF AROGENATE DEHYDROGENASES (ADHS) INVOLVED IN TYROSINE BIOSYNTHESIS IN TABLE BEET (BETA VULGARIS)
Samuel Lopez Nieves, Hiroshi Maeda.
University of Wisconsin-Madison, Madison, WI.
The aromatic amino acid tyrosine (Tyr) is a precursor of important secondary metabolites in plants (i.e., alkaloids, betalains, vitamins, etc.). As animals are unable to synthesize Tyr, we must uptake Tyr from external sources such as plants. However, most of our knowledge of Tyr biosynthesis comes from microbial studies, and limited research has been conducted for the Tyr pathway in plants. Previous studies reported that Tyr can be produced via 2 different routes: the 4-hydroxyphenylpyruvate (4-HPP) and arogenate pathways, in which the decarboxylation reaction is catalyzed by prephenate dehydrogenase (PDH), and arogenate dehydrogenase (ADH), respectively. In this study we investigated possible ADH and PDH enzymes involved in Tyr biosynthesis using table beet (Beta vulgaris) as a model system, which produces high levels of nitrogen-containing Tyr-derived pigments called betalains. We have detected ADH activity in the root and leaf crude extracts of table beet. Using the recently published sugar beet genome and PCR-based cloning, we identified 2 genes homologous to previously-reported Arabidopsis ADH genes in table beet. The purified recombinant enzymes showed only ADH but not PDH activity in vitro. These results suggest that Tyr is mainly produced via the arogenate pathway in Beta vulgaris. Currently, we are conducting kinetics analysis of the ADH enzymes. Genetic suppression of the identified ADHs in table beet can further allow us to investigate how plants produce and regulate biosynthesis of Tyr and Tyr-derived plant natural products.