An alternative pathway contributes to phenylalanine biosynthesis in plants via a cytosolic tyrosine:phenylpyruvate aminotransferase

Abstract

Phenylalanine is a vital component of proteins in all living organisms, and in plants is a precursor for thousands of additional metabolites. Animals are incapable of synthesizing phenylalanine and must primarily obtain it directly or indirectly from plants. Although plants can synthesize phenylalanine in plastids through arogenate, the contribution of an alternative pathway via phenylpyruvate, as occurs in most microbes, has not been demonstrated. Here we show that plants also utilize a microbial-like phenylpyruvate pathway to produce phenylalanine, and flux through this route is increased when the entry point to the arogenate pathway is limiting. Unexpectedly, we find the plant phenylpyruvate pathway utilizes a cytosolic aminotransferase that links the coordinated catabolism of tyrosine to serve as the amino donor, thus interconnecting the extra-plastidial metabolism of these amino acids. This discovery uncovers another level of complexity in the plant aromatic amino acid regulatory network, unveiling new targets for metabolic engineering.

A Root-Expressed l-Phenylalanine:4-Hydroxyphenylpyruvate Aminotransferase Is Required for Tropane Alkaloid Biosynthesis in Atropa belladonna

“…An amino acid alignment of the predicted protein sequence of the Ab-ArAT proteins and selected homologs indicates that each contains a conserved catalytic lysine that corresponds to residue 270 in Ab-ArAT1, together with conserved residues required for the binding of the pyridoxal-59-phosphate cofactor (Supplemental Figure 3). Ph-PPY-AT was previously shown to be localized in the cytosol rather than the chloroplast (Yoo et al, 2013); similarly, the Ab-ArATs identified in this study are not predicted to be localized to any organelles and lack the typical transit peptides that are characteristic of chloroplast-localized proteins. Phylogenetic analysis of ArATs from Arabidopsis, Atropa, and other Solanaceae, including the fully sequenced tomato and potato genomes, indicated an expansion of the ArAT family in Solanaceae compared with Arabidopsis (Figure 4; Supplemental Table 1) and suggested potential orthologous relationships.…”

“…However, plant genomes have retained multiple ArATs that likely utilize L-Phe and L-Tyr as substrates but, like Ab-ArAT4, are not predicted to be plastid localized. Recently, a petunia ArAT, Ph-PPY-AT, was shown to be localized to the cytoplasm and able to directly catalyze the formation of L-Phe from phenylpyruvate using L-Tyr as the amino donor, yielding 4-HPP as a by-product (Yoo et al, 2013). This reaction is analogous to the formation of L-Phe in bacteria and demonstrates the existence of a second, direct, extraplastidic route to L-Phe biosynthesis in plants and led to a proposed role for Ph-PPY-AT in modulating aromatic amino acid homeostasis (Whitaker et al, 1981;Fischer et al, 1993;Yoo et al, 2013).…”

“…Recently, a petunia ArAT, Ph-PPY-AT, was shown to be localized to the cytoplasm and able to directly catalyze the formation of L-Phe from phenylpyruvate using L-Tyr as the amino donor, yielding 4-HPP as a by-product (Yoo et al, 2013). This reaction is analogous to the formation of L-Phe in bacteria and demonstrates the existence of a second, direct, extraplastidic route to L-Phe biosynthesis in plants and led to a proposed role for Ph-PPY-AT in modulating aromatic amino acid homeostasis (Whitaker et al, 1981;Fischer et al, 1993;Yoo et al, 2013). In contrast, our data support a direct role for Ab-ArAT4 only in specialized metabolism, as silencing of this gene reduces tropane alkaloid accumulation but does not affect the abundance of the aromatic amino acids ( Figure 5; Supplemental Table 3).…”

“…Full-length transcripts were obtained for Ab-ArAT1 to 5, which encode proteins of between 419 and 441 amino acids (46 to 48 kD) that share between 50 and 72% amino acid identity. The majority of putative plant ArATs remain uncharacterized but the closely related Arabidopsis proteins encoded by At5g36160 and At5g53970, Ps-TyrAT from Papaver somniferum, Ph-PPY-AT from petunia, and Cm-ArAT1 from melon (Cucumis melo) all utilize L-Phe and L-Tyr as amino donors (Prabhu and Hudson, 2010;Lee and Facchini, 2011;Riewe et al, 2012;Yoo et al, 2013). An amino acid alignment of the predicted protein sequence of the Ab-ArAT proteins and selected homologs indicates that each contains a conserved catalytic lysine that corresponds to residue 270 in Ab-ArAT1, together with conserved residues required for the binding of the pyridoxal-59-phosphate cofactor (Supplemental Figure 3).…”

“…More recently, Ph-PPY-AT and Cm-ArAT1 were shown to preferentially catalyze the formation of L-Phe using L-Tyr as the amino donor and phenylpyruvate as the amino acceptor (Yoo et al, 2013). The kinetic parameters of purified recombinant Ab-ArAT4 were assessed using the three aromatic amino acids as amino donors together with several potential oxoacid acceptors as cosubstrates (Table 5).…”