Structure of Arabidopsis thaliana 5-methylthioribose kinase reveals a more occluded active site than its bacterial homolog

Abstract

Background: Metabolic variations exist between the methionine salvage pathway of humans and a number of plants and microbial pathogens. 5-Methylthioribose (MTR) kinase is a key enzyme required for methionine salvage in plants and many bacteria. The absence of a mammalian homolog suggests that MTR kinase is a good target for the design of specific herbicides or antibiotics.

Results: The structure of Arabidopsis thaliana MTR kinase co-crystallized with ATPgammaS and MTR has been determined at 1.9 A resolution. The structure is similar to B. subtilis MTR kinase and has the same protein kinase fold observed in other evolutionarily related protein kinase-like phosphotransferases. The active site is comparable between the two enzymes with the DXE-motif coordinating the nucleotide-Mg, the D238 of the HGD catalytic loop polarizing the MTR O1 oxygen, and the RR-motif interacting with the substrate MTR. Unlike its bacterial homolog, however, the Gly-rich loop (G-loop) of A. thaliana MTR kinase has an extended conformation, which shields most of the active site from solvent, a feature that resembles eukaryotic protein kinases more than the bacterial enzyme. The G- and W-loops of A. thaliana and B. subtilis MTR kinase adopt different conformations despite high sequence similarity. The ATPgammaS analog was hydrolyzed during the co-crystallization procedure, resulting in ADP in the active site. This suggests that the A. thaliana enzyme, like its bacterial homolog, may have significant ATPase activity in the absence of MTR.

Conclusion: The structure of A. thaliana MTR kinase provides a template for structure-based design of agrochemicals, particularly herbicides whose effectiveness could be regulated by nutrient levels. Features of the MTR binding site offer an opportunity for a simple organic salt of an MTR analog to specifically inhibit MTR kinase.

Figure 1

Multiple sequence alignment of selected MTR kinase sequences mapping the secondary structural elements found in A. thaliana (top) and B. subtilis (bottom) MTR kinase structures. The protein sequences aligned are from Arabidopsis thaliana, Oryza sativa, Bradyrhizobium japonicum, Wolinella succinogenes, Treponema denticola, Klebsiella pneumoniae, Bacillus anthracis, Psychroflexus torquis and Bacillus subtilis. Their gene identifiers (gi) from the National Center for Biotechnology Information (NCBI) are also listed. The secondary structural elements were defined according to DSSP [48], numbered by the order of their appearance, and named to be consistent between the two enzymes. α helices are presented as curly lines and β strands by arrows. η stands for a 3₁₀ helix. According to DSSP, the A. thaliana enzyme does not have the η2 helix found in the B. subtilis enzyme. The multiple sequence alignment was performed using the program T-Coffee [49], and the figure prepared using ESPript [50]. Strictly conserved residues are denoted in white and framed in blue boxes with a red background; residues conserved in at least 70% of the sequences are denoted in red and framed in blue boxes with a white background. The G-loop, the W-loop, the Mg-binding DXE-motif, the HGD catalytic loop, and the MTR-binding RR-motif are framed in green boxes. Residues circled in purple are involved in dimer formation. The linker region connecting the N-lobe and C-lobe is also indicated.

Figure 2

Structure of A. thaliana MTR kinase. (a) Stereo representation of the plant MTR kinase monomer. Monomer A of the MTRK-ADP-MTR complex is shown, with the nucleotide and substrate omitted. α helices are represented as grey coils with the 3₁₀ helices as wheat coils. β strands are represented as blue arrows and loops as grey tubes. The G-loop and W-loop are coloured in green. (b) Dimeric structure of the complex with monomer A coloured as in (a) and monomer B in green. (c) Stereo representation of detailed interactions between the two monomers. Figure 2 was prepared using PyMOL [51].

Figure 3

(a) Stereo representation of C^α superimposition of B. subtilis MTR kinase AMPPCP-MTR complex (PDB: 2PUN) in violet and A. thaliana MTR kinase ADP-MTR complex (PDB: 2PYW) in green. (b) Stereo stick presentation of the active sites of the B. subtilis MTR kinase in violet and A. thaliana MTR kinase in green. The two structures have been superimposed as in (a) and are shown in transparent cartoon. Monomer A is shown. The nucleotides and substrate MTR are shown in transparent stick representation (and labelled) in order to show the residues behind. This figure was prepared using PyMOL [51].

Figure 4

The active site of A. thaliana MTR kinase. (a) Stereo representation of the active site. The product ADP and the substrate MTR and nearby interacting residues are drawn in stick representation with C, N, O, P and S atoms coloured in cyan, blue, red, magenta, and orange, respectively. Hydrophobic residues are shown with their C atoms coloured in wheat. Residues that do not directly interact with ADP or MTR but are discussed in the text are shown with their C atoms coloured in green. The magnesium ions, chloride ion and water molecules are shown as yellow, violet and blue spheres, respectively. The σ_A-weighted Fo-Fc ligand (and water) omit map in blue mesh was calculated using CCP4 [52] and is contoured at 3σ. (b) Schematic diagram showing the hydrophilic interactions less than 3 Å between the ligands and the protein. The colour schemes of the atoms are as described in Panel (a). The distances in Å between the ligands and the protein are for monomer A while those for monomer B are in parentheses. If the two distances are the same, only one value is shown. The distances around the two octahedrally coordinated Mg(II) ions are labelled in orange. Residues from the G-loop are labelled in green. The distance between the N^δ amide nitrogen of N46 in the G-loop and the O4 of MTR is greater than 3 Å and hence is shown in grey instead of black. Both panels were prepared using PyMOL [51].

Figure 5

Comparison of the nucleotide binding pockets and loop conformations. Stereo surface representation of the A. thaliana MTR kinase ADP-MTR complex and B. subtilis MTR kinase AMPPCP-MTR complex are shown in panel (a) and (c), respectively with the G- and W-loop coloured in green and the ligands shown as purple sticks. Stereo cartoon representations of the four functionally important loops, the G-loop, the W-loop, the Mg-binding DXE-motif, and the HGD catalytic loop, found in the A. thaliana and B. subtilis enzymes are shown in (b) and (d), respectively. Substrates and important residues discussed in the text are shown in stick presentation with the same colour scheme as in Figure 4b. Residues labelled with an asterisk indicate that disordered side chains are observed in at least one subunit of all known structures of the B. subtilis enzyme. This figure was prepared using PyMOL [51].