Tyrosine metabolism: identification of a key residue in the acquisition of prephenate aminotransferase activity by 1β aspartate aminotransferase

Abstract

Alternative routes for the post‐chorismate branch of the biosynthetic pathway leading to tyrosine exist, the 4‐hydroxyphenylpyruvate or the arogenate route. The arogenate route involves the transamination of prephenate into arogenate. In a previous study, we found that, depending on the microorganisms possessing the arogenate route, three different aminotransferases evolved to perform prephenate transamination, that is, 1β aspartate aminotransferase (1β AAT), N‐succinyl‐l,l‐diaminopimelate aminotransferase, and branched‐chain aminotransferase. The present work aimed at identifying molecular determinant(s) of 1β AAT prephenate aminotransferase (PAT) activity. To that purpose, we conducted X‐ray crystal structure analysis of two PAT competent 1β AAT from Arabidopsis thaliana and Rhizobium meliloti and one PAT incompetent 1β AAT from R. meliloti. This structural analysis supported by site‐directed mutagenesis, modeling, and molecular dynamics simulations allowed us to identify a molecular determinant of PAT activity in the flexible N‐terminal loop of 1β AAT. Our data reveal that a Lys/Arg/Gln residue in position 12 in the sequence (numbering according to Thermus thermophilus 1β AAT), present only in PAT competent enzymes, could interact with the 4‐hydroxyl group of the prephenate substrate, and thus may have a central role in the acquisition of PAT activity by 1β AAT.

Harnessing evolutionary diversification of primary metabolism for plant synthetic biology

“…Mutating these two residues converted Arabidopsis PPA-AT to a general aromatic amino acid aminotransferase having broad substrate specificity (137). X-ray crystal structure analyses of plant and bacterial PPA-ATs further revealed the molecular basis of prephenate substrate recognition and identified two additional residues that further enhance prephenate specificity (140,201). Thus, these residues likely played key roles in the evolution of PPA-ATs that allow plants to synthesize Phe and tyrosine via the arogenate pathway.…”

“…in actinobacteria); and branched-chain aminotransferase (e.g. in cyanobacteria) (135,137,140). Notably, plant PPA-ATs are most closely related to the Ib aspartate aminotransferase-type of Chlorobi/Bacteroidetes (135,137).…”

“…PPA-ATs catalyze the committed step of the arogenate pathway of Phe and tyrosine biosynthesis (Fig. 3) (124 -127, 139) and are found in plants and some microbes (135,137,140). Biochemical characterization of PPA-AT homologs from various plants and microbes determined the phylogenetic distribution of their functional orthologs that are capable of transaminating prephenate (137, 140).…”

Evolutionary Diversification of Primary Metabolism and Its Contribution to Plant Chemical Diversity

“…Evolutionary analyses of the PPA-AT and ADT enzymes suggested that an ancestor of green algae and land plants appear to have acquired both of these two enzymes from an ancestor of Chlorobi/Bacteroidetes bacteria, likely through horizontal gene transfer (Dornfeld et al, 2014). Some cyanobacteria also have PPA-AT enzymes but with a distinct evolutionary origin from those of plants and Chlorobi/Bacteroidetes bacteria (Graindorge et al, 2014; Giustini et al, 2019). Thus, these dual primary metabolic pathways of isoprenoid and phenylalanine biosynthesis appear to have evolved in a common ancestor of Plantae.…”

Accumulation of the Auxin Precursor Indole-3-Acetamide Curtails Growth through the Repression of Cell Proliferation and Development-Related Transcriptional Networks

“…At the protein level (Figure 2b), the RTY1-1 point mutation translates into a P213S amino acid exchange [29], which is likely to cause major structural changes in the RTY enzyme. Protein modelling of RTY and RTY1-1 against the 1.7 Å crystal structure of a closely related prephenate aminotransferase from Arabidopsis [30] (PDB: 6F5V) and subsequent structure predictions suggested the loss of one -helix motif in RTY1-1 (13 -helices) relative to the wild-type protein (14 -helices) in the C-terminal part of the protein. Against our initial expectations, the ami1/rty double mutants still showed an auxin overproduction-related phenotype (Supplementary Figure S1), suggestive of a remaining conversion of IAM to IAA.…”