The Sequence-Function Relationship of Arabidopsis AROGENATE DEHYDRATASES
Abstract
Phenylalanine (Phe) is an essential aromatic amino acid that can only be synthesized de novo by microorganisms and plants. In microorganisms, Phe is synthesized through the prephenate pathway, requiring the activity of a prephenate dehydratase (PDT). In planta, Phe is synthesized instead through the arogenate pathway, requiring the enzyme arogenate dehydratase (ADT). In Arabidopsis, there is a family of six ADTs, named ADT1 through ADT6, that catalyze this step of Phe biosynthesis. All six AtADTs have a high sequence similarity and localize to the stromules of chloroplasts, the site of Phe biosynthesis in plants. Of note, two of the AtADTs, AtADT1 and AtADT2, can also act as PDTs, while AtADT5 has an alternate subcellular localization to the nucleus. As all six AtADTs are similar in sequence, it is difficult to predict what sequence is responsible for the distinct activities of AtADTs. Here, I identified candidate amino acids by detailed sequence analysis, and introduced targeted mutations generating amino acid substitutions. I employed the Δpha2 complementation assay, and established a novel test of ADT activity, to determine how these amino acid changes affect ADT and PDT activity of AtADTs. Using yeast-two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) analyses I analyzed how the putative nuclear localization of AtADT5 was affected. In both scenarios, only a few amino acids determined these unique functions. My work demonstrates that even a single amino acid change can affect enzymatic activity and localization and contributes to our knowledge of the sequence-function relationship of proteins and the divergence of gene family members.