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

Abstract

Aromatic amino acids are protein building blocks and precursors to a number of plant natural products, such as the structural polymer lignin and a variety of medicinally relevant compounds. Plants make tyrosine and phenylalanine by a different pathway from many microbes; this pathway requires prephenate aminotransferase (PAT) as the key enzyme. Prephenate aminotransferase produces arogenate, the unique and immediate precursor for both tyrosine and phenylalanine in plants, and also has aspartate aminotransferase (AAT) activity. The molecular mechanisms governing the substrate specificity and activation or inhibition of PAT are currently unknown. Here we present the X-ray crystal structures of the wild-type and various mutants of PAT from Arabidopsis thaliana (AtPAT). Steady-state kinetic and ligand-binding analyses identified key residues, such as Glu108, that are involved in both keto acid and amino acid substrate specificities and probably contributed to the evolution of PAT activity among class Ib AAT enzymes. Structures of AtPAT mutants co-crystallized with either α-ketoglutarate or pyridoxamine 5'-phosphate and glutamate further define the molecular mechanisms underlying recognition of keto acid and amino acid substrates. Furthermore, cysteine was identified as an inhibitor of PAT from A. thaliana and Antirrhinum majus plants as well as the bacterium Chlorobium tepidum, uncovering a potential new effector of PAT.

Structure-function analysis of interallelic complementation inROOTYtransheterozygotes

“…In fact, RTY belongs to a large family of C-S lyases/transaminases/alliinases several of which have been shown to function as dimers or tetramers (Nock and Mazelis, 1987; John, 1995; Breitinger et al, 2001). The closest related Arabidopsis protein in the database for which the crystal structure is available, At2g22250, an aspartate/prephenate aminotransferase MEE17, is known to form dimers (Holland et al, 2018). Consistent with the possibility of transheterozygote complementation, the amino acid substitutions in rty and TG8B map to different domains of the protein (see below), suggesting that they may compromise different activities.…”

“…We next employed molecular modeling to provide structural reasoning for the ability of transheterozygous mutations in RTY to restore the RTY activity and thus lead to a phenotype that is milder than either of the homozygous parents. A three-dimensional (3D) structural model of dimeric RTY was generated using the crystal structure of the closest RTY homolog from Arabidopsis thaliana , the aforementioned aminotransferase MEE17 (PDB code: 5WMH) (Holland et al, 2018) (Supplemental Fig. S4).…”

“…To our knowledge, no protein structure is currently available for RTY from Arabidopsis thaliana , but several related proteins have been structurally characterized through crystallization. Although the RTY protein sequence aligns with tyrosine and other aromatic amino acid aminotransferases from mouse, human, and bacterial proteomes, we chose the sequence of MEE17 (PDB 5WMH) from Arabidopsis thaliana , a bifunctional aspartate aminotransferase and glutamate/aspartate-prephenate aminotransferase (Holland et al, 2018), as the template for our analysis. MEE17 is a far better template for this molecular modeling study, as it is derived from the same species as RTY, harbors PLP in the binding pocket, includes an extra alpha helical structure that is lacking in the related human tyrosine aminotransferase (PDB 3DYD) (Supplemental Fig.…”

“…MODELLER v9.18 was used to construct a model of the dimeric RTY wild-type and transheterozygotes using Arabidopsis thaliana prephenate aminotransferase (PDB: 5WMH; Holland et al, 2018). During the model building process, we employed an optimization method involving conjugate gradients and molecular dynamics to minimize violations of the spatial restraints.…”

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

“…Most of the residues observed in the Ath‐PAT catalytic site are conserved in Rme‐PAT, Rme‐AAT, and Tth‐PAT. From structure comparison with Tth‐PAT, residues from the first α‐helix (Lys33(A), Pro34(A), Ser35(A), Lys36(A), Thr37(A), Met38(A) in Ath‐PAT ) are also involved in the closing of the catalytic site upon ligand binding. Lys259 of Ath‐PAT is covalently bound to PLP through an aldimine link.…”