Structure of Arabidopsis thaliana 5-methylthioribose kinase reveals a more occluded active site than its bacterial homolog
© Ku et al; licensee BioMed Central Ltd. 2007
Received: 04 July 2007
Accepted: 25 October 2007
Published: 25 October 2007
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.
The structure of Arabidopsis thaliana MTR kinase co-crystallized with ATPγS and MTR has been determined at 1.9 Å 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 ATPγS 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.
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.
S-adenosyl-L-methionine (SAM) is an important metabolite in plants as it not only provides the methyl group required for SAM-dependent biomethylation reactions, but also acts as the precursor for the biosynthesis of polyamines, nicotianamine, phytosiderophores, and the plant hormone ethylene . SAM is metabolized to S-adenosylhomocysteine (SAH) in biological methylation, and to 5'-methylthioadenosine (MTA) in many other essential biological processes. While SAH is the product and a potent inhibitor of all SAM-dependent methyltransferases , MTA inhibits spermine synthase  in polyamine biosynthesis, and 1-aminocyclopropane-1-carboxylic acid synthase  in ethylene biosynthesis. Both polyamine and ethylene are important in plant development, growth, stress response and survival [5–7]. To promptly remove the toxic by-product MTA and to ensure sufficient supply of methionine and SAM, a methionine salvage pathway that recycles the methylthio moiety of MTA to methionine has evolved in almost all organisms. In plants, this pathway is also known as the Yang cycle . In this cycle MTA is first depurinated to 5-methylthioribose (MTR), and then phosphorylated to form MTR 1-phosphate (MTR 1-P). MTR 1-P then undergoes isomerization, dehydration, enolization and dephosphorylation to form the acireductone, 1,2-dihydroxyl-3-keto-5-methylthiopentene. This acireductone is subsequently converted to 2-keto-4-methylthiobutyrate and then transaminated to methionine [8, 9].
Metabolic variation in how MTA is recycled to methionine exists between different species. In bacteria and plant cells, two enzymes, MTA nucleosidase and MTR kinase, are required to convert MTA to MTR-1-P while in mammalian cells, MTA phosphorylase  phosphorylizes MTA to MTR 1-P in a single step. This metabolic difference has been explored and analogs of MTA and MTR have been synthesized for the development of specific antibiotics and herbicides [11–14]. MTR kinase is not an essential enzyme for the organisms' survival if methionine is present in the growth medium , but in the absence of methionine, expression of MTR kinase will prompt an organism to uptake MTA and/or MTR from the environment to replenish its sulfur pool [15, 16]. Interestingly, even when sulfur is not depleted in the environment, numerous analogs of MTR have demonstrated selective growth inhibition of MTR kinase-containing bacteria [11, 12]. Not surprisingly, the bactericidal activity of these MTR analogs, particularly trifluoromethylthioribose, is enhanced if the de novo methionine synthesis pathway is also blocked . In plants, the expression of MTR kinase is also up-regulated under sulfur-limiting conditions, although the expression is not correlated with ethylene biosynthesis [15, 16, 18]. While most studies of MTR analogs have been performed on bacterial MTR kinases for the design of novel antibiotics, similar principles can be applied to the plant system to develop new herbicide and/or herbicide-resistant transgenic crops that would improve agriculture efficiency.
The modern rational approach to new agrochemical discovery includes structure-based design approaches , which requires a detailed understanding of the target enzyme's structure, catalytic mechanism and substrate specificity. To this end, we present the first structural analysis of A. thaliana MTR kinase in complex with ADP and MTR.
Results and Discussion
Overall fold of MTR kinase
The structure of A. thaliana MTR kinase has two subunits in the asymmetric unit related by a non-crystallographic 2-fold (Fig. 2b). Approximately 1300 Å2 or 6.7% of the surface area of each monomer is buried at the dimer interface . Even though the plant enzyme underwent a complex multi-step refolding process , its dimeric structure resembles that of the bacterial enzyme . Size exclusion chromatography of A. thaliana MTR kinase (data not shown), the physical characterization of other plant MTR kinases  as well as the apparent cooperative kinetics observed for the rice enzyme  all suggest that MTR kinase functions as a homodimer. The inter-subunit interactions in the dimer interface of A. thaliana MTR kinase are predominantly symmetrical and are concentrated at the C-terminus of helix α8 (Residues K217, K224, E229, R230 and A231). Additional inter-subunit interactions are found at the N-terminus of helix α5 (T161), the C-terminus of helix α6 (R179 and E182), as well as the loop between helices α10 and α11 (E327, I333 and Y334) (Fig. 1). The interactions between these residues are depicted in Fig. 2c. Interestingly, when comparing the dimeric interfaces of the B. subtilis and A. thaliana enzymes, the identities of the residues involved in dimerization are not conserved (Fig. 1). The interactions are nevertheless concentrated in approximately the same region of the protein, especially the C-terminal end of helix α8, and result in exactly the same dimeric structure.
Although the tertiary structures of MTR kinase and other protein kinase-like phosphotransferases are highly similar, their quaternary structures are distinct. While MTR kinase dimerizes mainly via interactions between helices α8, choline kinase dimerizes via interactions between helices that are structurally equivalent to helices α2 of MTR kinase. Although crystal structures of APH show the protein as dimers [28, 29], they are not physiologically relevant as the enzyme is observed as a monomer in solution . Rio2 protein kinase is also observed as a monomer .
Nucleotide binding site
Functionally important loops
There are four conserved loop regions in MTR kinases: the G-loop between strands β1 and β2, the HGD catalytic loop between strand β7 and the 310 helix η3, the Mg(II) binding DXE-motif between strands β9 and β10, and the semi-conserved W-loop between strand β3 and helix α2 (Fig. 1). The G-loop of MTR kinases has a highly conserved GXGNXN motif (residues 43–48) and is structurally analogous the "nucleotide positioning loop" in APH(3')-IIIa  and the "Gly triad" GXGXXG motif found in many eukaryotic protein kinases.
Gly-rich loop (G-loop) and solvent accessibility
DXE-motif and Mg binding
In the A. thaliana MTR kinase structure, two Mg(II) ions assist in the binding of the ADP. Mg(II) ions are important for MTR kinase activity. The kinase activity is optimal in the presence of Mg(II) ions, attenuated to 20% in the presence of Mn(II) ions, and abolished in the presence of Ca(II) ions . Residues D255 and E257 are part of the strictly conserved DXE-motif on the loop between strands β9 and β10 and are essential for Mg(II) binding. The first magnesium ion, Mg(1), is chelated by the carboxyl Oδ oxygens of D255, the carboxyl Oε oxygen of E257, the O2 oxygen of the β-phosphoryl group of ADP, and two water molecules. These water molecules in turn interact with the O1 oxygen of MTR and the catalytic residue D238 (see below) (Fig. 4b). The second magnesium ion, Mg(2), is chelated by the α- and β-phosphoryl oxygens of ADP, the Oδ oxygen of D255 and three water molecules. One of the water molecules that interacts with Mg(2) (W3 in monomer A and W6 in monomer B) also interacts with O1 oxygen of MTR (Fig. 4b). Unlike the bacterial MTR kinase where the water molecules surrounding the Mg(II) ions have poor electron density, both Mg(II) ions in the A. thaliana ADP-enzyme complex are coordinated in the most energetically favorable octahedral geometry.
Examination of amino acid sequences reveals that for all MTR kinases, except those from Nocardioides, Streptomyces and Clostridium species, the DXE-motif can be extended to a longer IDXEF motif (Fig. 1). The DXE-motif is always followed by a phenylalanine, F258 in A. thaliana MTR kinase, which is involved in MTR binding (see below). The significance of the DXE-motif is that this consensus sequence is analogous to the well defined DFG-motif that marks the start of the "activation loop" in protein kinases . Unlike other phosphotransferases such as APH(3')-IIIa or cAMP-dependent protein kinase (PKA), which use the aspartate in the DFG-motif (e.g. D208 in APH(3')-IIIa and D184 in PKA) and a second asparagine (e.g. N195 in APH(3')-IIIa and N171 in PKA) from the C-lobe to chelate the divalent metal ions, MTR kinase uses both acidic residues in the DXE-motif to bind Mg(II).
RR-motif in the MTR binding site
The substrate MTR is located beneath the nucleotide in the active site. In A. thaliana MTR kinase, MTR is bound in the higher energy α-ribofuranose anomer with the substrate's O1 hydroxyl cis to the O2 and O3 hydroxyl groups (Fig. 4a). MTR is also observed in the same α-anomer in the bacterial enzyme-substrate complex  (Fig. 3b). In the plant enzyme, the α-conformation is stabilized by interactions with D238 and R365 as well as F258, which interacts with the substrates hydrophobic face (Fig. 4b). The O1 and O2 oxygens of MTR form hydrogen bonds (~2.7 Å) with the carboxyl oxygens of D238 of the strictly conserved HGD-motif; while the O2 and O3 oxygens of MTR forms heteronuclear hydrogen bonds (~2.9 Å) with the guanidium nitrogens of R365 of the strictly conserved twin arginine RR-motif (Fig. 1). The second arginine R366 does not interact directly with MTR but may facilitate the release of the anionic product, MTR 1-P. In the A. thaliana enzyme, a lysine (K362) is located near the RR-motif (Fig. 4a), and to mitigate the electrostatic repulsion between these three positively charged residues, a chloride ion is found interacting with K362.
Interestingly, examination of the multiple sequence alignment (Fig. 1) shows that the residue equivalent to K362 in the A. thaliana enzyme, three residues upstream of the RR-motif, is always either a lysine or a glutamate residue in all known sequences of MTR kinases. Most bacterial MTR kinases including the well-studied Klebsiella pneumoniae and all of the Bacillus enzymes, have a glutamate at this position. This glutamate will help neutralize the positive charge imposed by the RR-motif in the MTR binding site. A lysine residue near the RR-motif, as found in the plant A. thaliana, Oryza sativa (rice), Vitis vinifera (grape), and nitrogen-fixing bacteria Bradyrhizobium japonicum and Wolinella succinogenes, would perturb MTR binding by charge repulsion when the pH falls below the pKa of lysine. Thus, the K362 in A. thaliana MTR kinase could potentially explain why this enzyme's in vitro activity is optimal at pH 9, near the pKa of lysine, while the optimal activity for K. pneumoniae MTR kinase occurs at pH 7 (Reference  and see Additional file 1).
W-loop and MTR binding specificity
The W-loop between strand β3 and helix α2, named after the semi-conserved tryptophan (W) at the tip of this loop, appears to modulate the size of chemical group at the 5-position that can fit into the MTR binding site. In A. thaliana MTR kinase, the 5-methylthio moiety of the MTR appears to be capped by W76 of the W-loop and residues V184 and V370 from the C-lobe (Fig. 4a). These three residues would appear to restrict the length of the alkylthio moiety at the 5-position. Although these three residues are not strictly conserved across MTR kinase sequences, they are structurally and functionally equivalent to residues W74 of the W-loop, and L180 and L345 in B. subtilis MTR kinase. However, many MTR analogs with bulky extensions at the 5-position have been shown to be substrates and/or inhibitors of MTR kinase [11, 35], although the bulkiness of the substitution does not correlate with their inhibitory concentrations . In fact, these inhibitors have comparable Km values (in the μM range) to the native substrate in many bacterial and plant MTR kinases [15, 32, 33]. Moreover, comparison between A. thaliana and B. subtilis MTR kinase shows that both the 5-methylthio moiety of MTR and the W-loop adopt different conformations in the otherwise comparable overall active sites (Fig. 3b and 5). These results suggest that the W-loop needs to be flexible to allow the entry of different sized MTR analogs to the active site. This hypothesis is consistent with the observation in B. subtilis MTR kinase structures that the density of the W-loop is often disordered , implying its flexibility.
In the structural alignment of bacterial and plant MTR kinase, the bound ADP in A. thaliana is shifted toward the C-lobe when compared to the nucleotide in the B. subtilis enzyme (Fig. 3b). This observation raises the question whether the plant MTR kinase has a more closed conformation between the two-lobes than its bacterial homolog. Although the overall structural alignment of the two enzymes shows no major conformation changes, a minor rigid body shift of 1–1.5 Å in the N-lobe becomes apparent when only the C-lobes of the two structures are aligned. However, this displacement does not definitively demonstrate a potential inter-lobal movement in MTR kinase on substrate binding, because the intrinsic variation resulting from the sequence variation between the two enzymes – even neglecting the intrinsic structure coordinate errors of both structures – is more than 1.5 Å. In other words, a 1.5 Å displacement observed between the N-lobes is within the expected structural variation of two non-identical protein sequences .
Despite the low overall Cα RMSD between the structures of A. thaliana and B. subtilis MTR kinase, the conformations of the G- and W-loop in the two enzymes differ by ~3.8 Å and 5.3 Å in their Cα RMSDs, respectively (Fig. 5b and 5d). This observation is striking as the sequence identity of the 6-residue G-loop between the two enzymes is 100%, and that of the 16-residue W loop is 50% (Fig. 1). These RMSD values are close to the expected values if two random loops of the same length are taken from the Protein Data Bank and structurally aligned . The conformational variations between the two enzymes are the result of different interactions outside the loop regions. In the A. thaliana enzyme, the highly conserved E41, preceding the G-loop, interacts with R70 of the W-loop via an ionic interaction (Fig. 5b). The corresponding E37 and K68 in the B. subtilis enzyme do not interact (Fig. 5d). Moreover, in the plant enzyme, the main chain carbonyl oxygen of N46 (G-loop) forms a hydrogen bond to the main chain nitrogen of C71 (W-loop) (Fig. 5b), but in the bacterial enzyme, the corresponding N42 (G-loop) interacts with main chain carbonyl oxygen of V70 (W-loop) via the side chain Nδ nitrogen instead (Fig. 5d). The difference in loop conformations could also be due to the differences in how they interact with the substrate in the homologous active sites (Fig. 3b). Until an apo-form of A. thaliana MTR kinase becomes available, it would be difficult to determine whether the variations in the loop conformations are substrate induced.
ATPase activity in MTR kinase
The A. thaliana MTR kinase complex was prepared by co-crystallization with excess but equal amount of ATPγS and MTR, but surprisingly, ADP and MTR were found in the active site. Thus, consistent with the soaking and co-crystallization experiments of bacterial MTRK in the presence of ATP , the plant enzyme appears to be able to hydrolyze the weakly hydrolysable ATPγS to ADP. Without extensive kinetic characterization it is hard to know whether this ATP hydrolysis is coupled to phosphorylation of MTR, because while only MTR was found in the active site, it might bind preferentially to MTR 1-P. The observation that A. thaliana MTR kinase, like its bacterial homologue, may have considerable ATPase activity in the absence of the substrate MTR will have an impact on the strategies used to design herbicides that target the enzyme.
The structure of a eukaryotic MTR kinase has been determined for the first time. Although the enzyme has been refolded into its active state, it has comparable tertiary and quaternary structures to its bacterial homolog. 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 MTR. The G-loop and W-loop of the A. thaliana MTR kinase adopt different conformations, a consequence potentially of differences in their microenvironments. Unlike its bacterial homolog, the G-loop of A. thaliana MTR kinase has an extended conformation, which shields most of the active site from solvent, a feature that resembles a eukaryotic protein kinase more closely than the bacterial enzyme. The A. thaliana MTR kinase has optimal activity at higher pH than its bacterial homolog and this can be explained by the presence a lysine, rather than a glutamate, near the RR-motif in MTR binding site. The structure of A. thaliana MTR kinase also provides a template for structure-based design of agrochemicals – particularly herbicides whose effectiveness could be regulated by nutrient levels. Generic protein kinase inhibitors likely inhibit MTR kinase as they inhibit the remotely homologous APH enzymes , but these kinase inhibitors are often associated with complex steps of synthesis and isolation and high cost. The strictly conserved RR-motif in MTR binding site along with the nearby catalytic aspartate offers an opportunity for a simpler organic salt of an MTR analog to specifically inhibit MTR kinase at lower cost.
A. thaliana MTR kinase was cloned, and expressed in E. coli, and purified from inclusion bodies as a linear peptide, refolded into its active form and co-crystallized with substrates as described previously . The effect of pH on MTR kinase activity was examined using a modification of the standard enzyme assay  in which the imidazole buffer (pH 6.5) was replaced by 100 mM sodium phosphate (pH 4 – 9.5). Assays used 300 ng of recombinant enzyme and were performed in triplicate. Although prior to co-crystallization the enzyme was incubated with ATPγS and MTR, only ADP and MTR were found in the resulting structure. The crystals belong to space group C 2 (a = 162.5 Å, b = 83.2 Å, c = 91.1 Å, β = 117.8°) with a dimer per asymmetric unit. Prior to data collection the crystal was soaked in a solution containing 25% (v/v) ethylene glycol, 7.5% (w/v) PEG8000, 75 mM sodium cacodylate pH 6.5, and 0.15 M magnesium acetate for 5 seconds and flash frozen in a nitrogen cryo-stream. Data were collected to 1.9 Å resolution at Station X8C, National Synchrotron Light Source, Brookhaven National Laboratory, and processed using d*TREK  and the DREAR program package .
Diffraction Data and Crystallographic Refinement Statistics
Cell dimensions (Å, °)
a = 162.5, b = 82.2, c = 91.1 β = 117.8
80.6 – 1.9
Number of reflections (working/test)
Number of protein atoms/heteroatomsc
Number of water molecules
RMS deviation from ideal values
Bond length (Å)
Bond angle (°)
Average B factor (Å2)
Ligands (ADP and MTR)
Total Favoured (%)
Total Allowed (%)
DPIf coordinate error based on Rfree (Å)
The coordinates of the A. thaliana MTR kinase in complex with ADP and MTR have been deposited in the Protein Data Bank, accession number 2PYW.
List of abbreviations
Protein Data Bank
- MTR 1-P:
3',5"-aminogylcoside phosphotransferase type IIIa
cyclic adenosine monophosphate
protein kinase A
root mean square deviation
flavin adenine dinucleotide
nicotinamide adenine dinucleotide (phosphate)
This work is supported by research grants from the Canada Institutes of Health Research (#43998), the United States Department of Agriculture (#02-0047), Idaho INBRE program (NIH NCRR P20RR016464), W.M. Keck Foundation and the Lucille P. Markey Charitable Trust. P.L.H. is the recipient of a Canada Research Chair. S.-Y.K. has been funded in part by postgraduate scholarships from the Natural Sciences and Engineering Research Council of Canada, the Ontario Student Opportunities Trust Fund and The Hospital for Sick Children Foundation Student Scholarship Program. Station X8-C is supported by the United States Department of Energy, and Multi-User Resource grants from the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada.
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