Doctor of Philosophy
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.
Summary for Lay Audience
As sessile organisms, plants produce a wide variety of molecules to survive in their environment. These molecules provide protection from environmental stresses, herbivory, and infection. These molecules, termed specialized metabolites, are also of importance in agriculture, as they are related to the hardiness and the nutritional quality of crop species. Many specialized metabolites are derived from one of three aromatic amino acids: tryptophan (Trp), tyrosine (Tyr) or phenylalanine (Phe). Understanding how these aromatic amino acids are synthesized is crucial for heartier, more nutritious crop production. The coordinated synthesis of these molecules is tightly regulated and robust, often involving entire gene families to allow for differential expression during development or in different plant organs. In Arabidopsis, the last step of Phe synthesis is performed by a family of six enzymes, called arogenate dehydratases (AtADTs). Protein families like the AtADTs provide an opportunity to study the similarities and differences between closely related proteins. All six AtADTs have the same function, and they all localize to the same place in the cell, the chloroplast. Unsurprisingly, as all six AtADTs perform the same role, their protein sequences (the composition and order of amino acids) are very similar. However, there are some notable differences between the AtADT family members. My work analyzes these supposedly small differences in protein sequence between AtADT family members to identify which sequences determine these differences in function. My study reveals that even very small changes at a single amino acid level, can alter protein function and localization within a cell. Thus, my work contributes to our understanding of the relationship between the amino acid sequence of proteins and their function.
Clayton, Emily J., "The Sequence-Function Relationship of Arabidopsis AROGENATE DEHYDRATASES" (2022). Electronic Thesis and Dissertation Repository. 8604.
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