Crystal Structure of the Chloroplastic Glutamine Phosphoribosylpyrophosphate Amidotransferase GPRAT2 From Arabidopsis thaliana

Abstract

Chloroplastic glutamine phosphoribosylpyrophosphate amidotransferase (GPRATase) catalyzes the first committed step of de novo purine biosynthesis in Arabidopsis thaliana, and DAS734 is a direct and specific inhibitor of AtGPRAT, with phytotoxic effects similar to the leaf beaching phenotypes of known AtGPRAT genetic mutants, especially cia1 and atd2. However, the structure of AtGPRAT and the inhibition mode of DAS734 still remain poorly understood. In this study, we solved the structure of AtGPRAT2, which revealed structural differences between AtGPRAT2 and bacterial enzymes. Kinetics assay demonstrated that DAS734 behaves as a competitive inhibitor for the substrate phosphoribosyl pyrophosphate (PRPP) of AtGPRAT2. Docking studies showed that DAS734 forms electrostatic interactions with R264 and hydrophobic interactions with several residues, which was verified by binding assays. Collectively, our study provides important insights into the inhibition mechanism of DAS734 to AtGPRAT2 and sheds light on future studies into further development of more potent herbicides targeting Arabidopsis GPRATases.

Keywords: Arabidopsis thaliana; DAS734; X-ray crystallography; chloroplastic glutamine phosphoribosylpyrophosphate amidotransferase; competitive inhibition; herbicide.

Figure 1

Overview of AtGPRAT2. (A) The domain architecture of AtGPRAT2, and the construct for crystallization is indicated in the bottom. (B) Cartoon model of the tetramer conformation of AtGPRAT2. The protomers are shown in different colors and the 4Fe-4S cofactor is shown in sticks. Two views are shown. (C) Cartoon model of one protomer of AtGPRAT2. The Glnase domain and PRTase domain are colored in green and cyan, respectively. Two views are shown.

Figure 2

Active site of AtGPRAT2. (A) Structural superimposition of AtGPRAT2, EcGPRAT in the active [Protein Data Bank (PDB): 1ECC] and inactive (PDB: 1ECF) conformation, and Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase (BsGPRAT) in the inactive conformation (PDB: 1GPH). Only the indicated loop regions are shown in cartoon models. The ligands in 1ECC are shown as sticks. cPRPP, carbocyclic phosphoribosyl pyrophosphate (PRPP). DON, 6-diazo-5-oxo-L-nor-leucine, is an analog of substrate glutamine. The conserved loop regions are marked. (B) An enlarged view of the active site of AtGPRAT2. Several active site residues (green) and the substrate analogs cPRPP and DON in the aligned structure of EcGPRAT in the active conformation (PDB: 1ECC) are shown in sticks. (C) Activity assay with the wildtype and several active site mutants of AtGPRAT2. Error bars are standard error of the mean (SEM) values of three independent experiments.

Figure 3

The inhibition mode of DAS734 on AtGPRAT2. (A) Inhibition kinetics showing that DAS734 is a competitive AtGPRAT2 inhibitor with respect to phosphoribosyl pyrophosphate (PRPP). The labels indicate different DAS734 concentrations. The data was fitted to the mixed inhibition model in GraphPad Prism software, and the alpha value of 12.56 calculated by this model indicated a competitive inhibition. Error bars are standard error of the mean (SEM) values of three independent experiments. (B) Structural superimposition of AtGPRAT2 with docked DAS734, EcGPRAT in the active [Protein Data Bank (PDB): 1ECC] and BsGPRAT in the inactive conformation (PDB: 1GPH). The cPRPP and 6-diazo-5-oxo-L-nor-leucine (DON) in the structure of 1ECC are shown as cyan sticks. The two AMP molecules in the A and C sites are shown as slate sticks. The docked DAS734 is shown in yellow sticks with the chlorine atom colored in gray.

Figure 4

The binding site of DAS734. (A) Structural superimposition of AtGPRAT2 with docked DAS734 and EcGPRAT in the active [Protein Data Bank (PDB): 1ECC] conformation. The AtGPRAT2 residues involved in DAS734 binding are shown as sticks. The carbocyclic phosphoribosyl pyrophosphate (cPRPP) and 6-diazo-5-oxo-L-nor-leucine (DON) molecules in EcGPRAT are colored as in Figure 3B. Electrostatic interactions and hydrogen bonds are shown as red dashed lines. (B) Interaction of AtGPRAT2 mutants and DAS734 as detected by isothermal titration calorimetry (ITC). The ITC curves are shown. The calculated K_d mean ± SE values are indicated. The top plots represent time, and the bottom plots represent molar ratio. These experiments were performed twice with equivalent results.