Characterisation of the first enzymes committed to lysine biosynthesis in Arabidopsis thaliana

Abstract

In plants, the lysine biosynthetic pathway is an attractive target for both the development of herbicides and increasing the nutritional value of crops given that lysine is a limiting amino acid in cereals. Dihydrodipicolinate synthase (DHDPS) and dihydrodipicolinate reductase (DHDPR) catalyse the first two committed steps of lysine biosynthesis. Here, we carry out for the first time a comprehensive characterisation of the structure and activity of both DHDPS and DHDPR from Arabidopsis thaliana. The A. thaliana DHDPS enzyme (At-DHDPS2) has similar activity to the bacterial form of the enzyme, but is more strongly allosterically inhibited by (S)-lysine. Structural studies of At-DHDPS2 show (S)-lysine bound at a cleft between two monomers, highlighting the allosteric site; however, unlike previous studies, binding is not accompanied by conformational changes, suggesting that binding may cause changes in protein dynamics rather than large conformation changes. DHDPR from A. thaliana (At-DHDPR2) has similar specificity for both NADH and NADPH during catalysis, and has tighter binding of substrate than has previously been reported. While all known bacterial DHDPR enzymes have a tetrameric structure, analytical ultracentrifugation, and scattering data unequivocally show that At-DHDPR2 exists as a dimer in solution. The exact arrangement of the dimeric protein is as yet unknown, but ab initio modelling of x-ray scattering data is consistent with an elongated structure in solution, which does not correspond to any of the possible dimeric pairings observed in the X-ray crystal structure of DHDPR from other organisms. This increased knowledge of the structure and function of plant lysine biosynthetic enzymes will aid future work aimed at improving primary production.

Figure 1. Comparison of bacterial and plant DHDPS.

Structures shown for E. coli (1yxc) and N. sylvestris .

Figure 2. Analytical ultracentrifugation of At-DHDPS2 and At-DHDPR2.

Sedimentation velocity analysis of At-DHDPS2 and At-DHDPR2. A) Continuous sedimentation coefficient distribution [(c)s] analysis of At-DHDPS2 at a concentration of 0.75 mg.mL⁻¹ (black line). The RMSD and Runs Test Z (RTZ) scores for the fit were 0.008 and 3.2 respectively. B) (c)s analysis of At-DHDPR2 at concentrations of 0.1 mg.mL⁻¹ (black line; RMSD = 0.009, RTZ = 2.4), 0.2 mg.mL⁻¹ (red line; RMSD = 0.010, RTZ = 2.0), 0.4 mg.mL⁻¹ (green line; RMSD = 0.014, RTZ = 8.6) 0.8 mg.mL⁻¹ (pink line; RMSD = 0.013, RTZ = 4.9) and 1.6 mg.mL⁻¹ (blue line; RMSD = 0.015, RTZ = 7.4). Radial absorbance data for the three lower protein concentrations were acquired at a different wavelength to those of the two highest protein concentrations, and the c(s) distributions were scaled accordingly. Residuals for the fits are shown in Figure S7.

Figure 3. Light scattering analysis of At-DHDPS2 and At-DHDPR2 At-DHDPS2 and At-DHDPR2 were loaded onto a P3000 column and eluted using 20 mM Tris.HCl, 150 mM NaCl.

Right angle light scattering and refractive index were measured and used to calculate the molecular weight, as described in the materials and methods. The dotted lines show the expected molecular weight calculated from the sequence for the dimeric and tetrameric enzymes.

Figure 4. Crystal structures of unliganded and lysine bound At-DHDPS2.

A) Wall-eyed stereo image of the Cα superposition of At-DHDPS2 with bound lysine (blue Cα trace) and unliganded At-DHDPS2 (gold Cα trace; rmsd = 0.3 Å). The lysine molecules bound at the allosteric site of each monomer of the tetramer are shown in yellow (stick representation). B) The lysine binding site at the monomer-monomer interface of the tight-dimer showing residues in contact with the bound lysine molecules (yellow). Electron density around the bound lysine (grey mesh, contoured at 1.0 sigma) was calculated using refined coordinates omitting the bound lysine molecules. Residues contributed by each monomer of the tight-dimer are shown in different shades of blue, and are indicated by the use of the prime (’) symbol. C) overlay of the lysine binding residues of the tight-dimer from the lysine bound (blue) and unliganded (gold) structures. Lysine molecules are shown in yellow. Residues contributed by each monomer of the tight-dimer are shown in different shades of blue or gold, and are indicated by the use of the prime (’) symbol.

Figure 5. X-Ray scattering data of At-DHDPS2.

Data were collected in the absence of ligand, or in the presence of 1 mM (S)-lysine, top panel; curves have been arbitrarily displaced along the logarithmic axis for clarity. Solid lines show the scattering profile from the unliganded crystal structure, calculated using CRYSOL. Distance-distribution functions, p(r) for the unbound and ligand bound At-DHDPS2 were determined using the indirect Fourier tranformation package GNOM (bottom panel).

Figure 6. X-Ray scattering of DHDPR.

Data were collected for At-DHDPR2, Ec-DHDPR and Tm-DHDPR (panel A); curves have been arbitrarily displaced along the logarithmic axis for clarity. Data was analysed using GNOM (fitted data shown by red line in panel A) to calculate a distance distribution function for each enzyme (panel B).

Figure 7. Results of ab initio modeling of At-DHDPR from SAXS data.

Models were generated using GASBOR (left panels) and DAMMIN (right panels). The structural homology model generated by SWISS-MODEL and fitted to the scattering data using CORAL is superimposed for comparison.