Investigating substrate specificity in arogenate versus prephenate dehydratases

BACKGROUND: Phenylalanine is an essential aromatic amino acid that can only be synthesized de novo by microorganisms and plants. In microorganisms, phenylalanine is synthesized through the prephenate pathway, requiring the activity of a prephenate dehydratase (PDT). In plants, phenylalanine is synthesized instead mostly through the arogenate pathway, requiring the enzyme arogenate dehydratase (ADT). In Arabidopsis, there is a family of six ADTs that catalyze this final step of phenylalanine biosynthesis. However, two of the AtADTs, AtADT1 and AtADT2, can also act as PDTs. All six AtADTs have a high sequence similarity, making it difficult to determine in silico which amino acids determine substrate specificity. RESULTS: Here, we use domain swapping, targeted mutagenesis, and pha2 yeast complementation to investigate amino acids that confer PDT activity in pha2 yeast. In addition, we established a novel in vivo test of ADT activity to determine how these amino acid changes affect ADT and PDT activity of the AtADTs. CONCLUSIONS: Our results demonstrate that a combination of amino acids in the regulatory ACT domain contributes to both ADT and PDT activity in the AtADTs.