Phenylalanine Biosynthesis in Arabidopsis thaliana

Abstract

There is much uncertainty as to whether plants use arogenate, phenylpyruvate, or both as obligatory intermediates in Phe biosynthesis, an essential dietary amino acid for humans. This is because both prephenate and arogenate have been reported to undergo decarboxylative dehydration in plants via the action of either arogenate (ADT) or prephenate (PDT) dehydratases; however, neither enzyme(s) nor encoding gene(s) have been isolated and/or functionally characterized. An in silico data mining approach was thus undertaken to attempt to identify the dehydratase(s) involved in Phe formation in Arabidopsis, based on sequence similarity of PDT-like and ACT-like domains in bacteria. This data mining approach suggested that there are six PDT-like homologues in Arabidopsis, whose phylogenetic analyses separated them into three distinct subgroups. All six genes were cloned and subsequently established to be expressed in all tissues examined. Each was then expressed as a Nus fusion recombinant protein in Escherichia coli, with their substrate specificities measured in vitro. Three of the resulting recombinant proteins, encoded by ADT1 (At1g11790), ADT2 (At3g07630), and ADT6 (At1g08250), more efficiently utilized arogenate than prephenate, whereas the remaining three, ADT3 (At2g27820), ADT4 (At3g44720), and ADT5 (At5g22630)

Subcellular localization of Arabidopsis arogenate dehydratases suggests novel and non-enzymatic roles

“…The N-terminal domain is unique to plant ADTs and is not found in the bacterial or fungal proteins. In A. thaliana ADTs, this domain is ~100–130 amino acids in length, and are likely to be chloroplast TPs according to sequence prediction programs, which is consistent with phenylalanine biosynthesis occurring in chloroplasts (Jung et al , 1986; Cho et al , 2007; Li and Chiu, 2010) and a chloroplastic localization identified for ADTs in protoplasts (Rippert et al , 2009). …”

“…(A) Phenylalanine can be synthesized in plants using either the prephenate (top) or the arogenate (bottom) pathway (Cho et al , 2007; Maeda and Dudareva, 2012). Prephenate is either decarboxylated/dehydrated to phenylpyruvate (PP) by a prephenate dehydratase (PDT) and PP is then transaminated by a phenylpyruvate aminotransferase (PPAT) to phenylalanine.…”

“…The Arabidopsis thaliana genome encodes a small gene family of six ADT genes, all sharing similar sequences and domain structures (Ehlting et al , 2005; Cho et al , 2007). Families of ADTs are common in plant genomes and have been identified in both monocot and dicot species (Tuskan et al , 2006; Yamada et al , 2008; Maeda et al , 2010, 2011).…”

“…For example, all six A. thaliana ADTs accept arogenate as a substrate, as their name suggests, but two of the six ADTs also accept prephenate [meaning they can act as ADTs and prephenate dehydratases (PDTs; Fig. 1A; Cho et al , 2007; Bross et al , 2011)]. Furthermore, ADTs differentially contribute to lignin content and bolting/flowering transition.…”

“…1B), a putative N-terminal transit peptide (TP), an internal catalytic domain, and a C-terminal ACT (aspartokinase–chorismate mutase–TyrA) domain (Cho et al , 2007). Both the catalytic and ACT domains are conserved across plant, bacterial, and fungal ADTs and PDTs, with the catalytic domain decarboxylating/dehydrating prephenate and/or arogenate (Cho et al , 2007; Bross et al , 2011) while the ACT domain is involved in allosteric regulation induced by ligand binding (Tan et al , 2008; Vivan et al , 2008). The N-terminal domain is unique to plant ADTs and is not found in the bacterial or fungal proteins.…”