Phylobiochemical Characterization of Class-Ib Aspartate/Prephenate Aminotransferases Reveals Evolution of the Plant Arogenate Phenylalanine Pathway

Abstract

The aromatic amino acid Phe is required for protein synthesis and serves as the precursor of abundant phenylpropanoid plant natural products. While Phe is synthesized from prephenate exclusively via a phenylpyruvate intermediate in model microbes, the alternative pathway via arogenate is predominant in plant Phe biosynthesis. However, the molecular and biochemical evolution of the plant arogenate pathway is currently unknown. Here, we conducted phylogenetically informed biochemical characterization of prephenate aminotransferases (PPA-ATs) that belong to class-Ib aspartate aminotransferases (AspAT Ibs) and catalyze the first committed step of the arogenate pathway in plants. Plant PPA-ATs and succeeding arogenate dehydratases (ADTs) were found to be most closely related to homologs from Chlorobi/Bacteroidetes bacteria. The Chlorobium tepidum PPA-AT and ADT homologs indeed efficiently converted prephenate and arogenate into arogenate and Phe, respectively. A subset of AspAT Ib enzymes exhibiting PPA-AT activity was further identified from both Plantae and prokaryotes and, together with site-directed mutagenesis, showed that Thr-84 and Lys-169 play key roles in specific recognition of dicarboxylic keto (prephenate) and amino (aspartate) acid substrates. The results suggest that, along with ADT, a gene encoding prephenate-specific PPA-AT was transferred from a Chlorobi/Bacteroidetes ancestor to a eukaryotic ancestor of Plantae, allowing efficient Phe and phenylpropanoid production via arogenate in plants today.

Relaxation of tyrosine pathway regulation underlies the evolution of betalain pigmentation in Caryophyllales

“…The His‐tagged recombinant protein expression was carried out as we described previously (Dornfeld et al ., ). For glutathione S‐transferase (GST)‐tagged recombinant protein expression, the cloned pGEX‐2T vectors were introduced into Rosetta‐2 E. coli competent cells (Novagen, Madison, WI, USA) and cultured overnight at 37°C, 200 rpm in 10 ml LB medium containing Ampicillin (100 μg ml −1 ).…”

Structural basis for substrate recognition and inhibition of prephenate aminotransferase from Arabidopsis

“…Earlier work suggests that the evolutionary basis for PAT acquisition in plants was lateral gene transfer from Chlorobi/Bacteroidetes (Dornfeld et al ., ). To test if cysteine also inhibited PAT homologs in other plants and in microbes, two previously characterized PATs from Antirrhinum majus (snapdragon) and Chlorobium tepidum (a Gram‐negative, thermophilic green sulfur bacterium) were assayed for potential inhibition (Figure a).…”

“…The T84V, A168G, K169V, K169S, R445G and T84V/K169V PAT mutants bound alanine to varying degrees, whereas wild‐type AtPAT did not bind alanine. In earlier work (Dornfeld et al ., ), the amino donor for AtPAT was changed from aspartate to tryptophan in the T84V/K169V double mutant. To analyze this specificity change further, the binding of tryptophan and tyrosine was assessed (Table ).…”

“…Previous biochemical studies of plant PATs revealed a higher specific activity with prephenate than with arogenate (Maeda et al ., ); however, detailed kinetic and ligand‐binding analyses of a plant PAT remain to be performed. A phylobiochemical investigation identified two residues (Thr84 and Lys169 in AtPAT) that were conserved among PATs but were absent in AATs (Dornfeld et al ., ). Corresponding residues are also critical for binding of dicarboxylates in the orthologous enzyme from Thermus thermophilus , which belongs to the class Ib AAT family (Nobe et al ., ; Nakai et al ., ; Ura et al ., ; Wang and Maeda, ).…”

“…Corresponding residues are also critical for binding of dicarboxylates in the orthologous enzyme from Thermus thermophilus , which belongs to the class Ib AAT family (Nobe et al ., ; Nakai et al ., ; Ura et al ., ; Wang and Maeda, ). The T84V and K169V mutants of AtPAT significantly reduce PAT activity (Dornfeld et al ., ). Interestingly, the T84V/K169V mutant lost function with prephenate but gained activity with alanine and tryptophan as amino donors and with 4‐HPP as the keto acid.…”