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To whom correspondence should be addressed: Structural Plant Biology Laboratory, Dept. of Botany and Plant Biology, Sciences III, 30 Quai E. Ansermet, University of Geneva, 1211 Geneva, Switzerland. Tel.: 41-22-379-3013.
* This work was supported by the European Research Council under the European Union's Seventh Framework Programme (FP/2007–2013)/ERC Grant Agreement 310856, the Max Planck Society, and the European Molecular Biology Organisation (EMBO) Young Investigator Programme (to MH). The authors declare that they have no conflicts of interest with the contents of this article.
Triphosphate tunnel metalloenzymes (TTMs) are present in all kingdoms of life and catalyze diverse enzymatic reactions such as mRNA capping, the cyclization of adenosine triphosphate, the hydrolysis of thiamine triphosphate, and the synthesis and breakdown of inorganic polyphosphates. TTMs have an unusual tunnel domain fold that harbors substrate- and metal co-factor binding sites. It is presently poorly understood how TTMs specifically sense different triphosphate-containing substrates and how catalysis occurs in the tunnel center. Here we describe substrate-bound structures of inorganic polyphosphatases from Arabidopsis and Escherichia coli, which reveal an unorthodox yet conserved mode of triphosphate and metal co-factor binding. We identify two metal binding sites in these enzymes, with one co-factor involved in substrate coordination and the other in catalysis. Structural comparisons with a substrate- and product-bound mammalian thiamine triphosphatase and with previously reported structures of mRNA capping enzymes, adenylate cyclases, and polyphosphate polymerases suggest that directionality of substrate binding defines TTM catalytic activity. Our work provides insight into the evolution and functional diversification of an ancient enzyme family.
). The catalytic core of VTC maps to a cytoplasmic 8-stranded β-tunnel domain in Vtc4p, which harbors binding sites for the nucleotide substrate, for a manganese metal co-factor and for an orthophosphate priming the polymerase reaction (
The Vtc4p β-tunnel domain is not unique to eukaryotic polyP polymerases but is a structural hallmark of triphosphate tunnel metalloenzymes (TTMs), whose characteristic features are the presence of a topologically closed hydrophilic β-barrel, the preference for triphosphate-containing substrates, and for a divalent metal co-factor (PFAM (
). However, the lack of substrate- and product-bound structures has made it difficult to define the sequence-fingerprints responsible for a specific enzyme activity in individual family members.
Protein Expression and Purification
AtTTM3 (Uniprot ID Q9SIY3) was cloned into vector pMH-HT providing an N-terminal His6 tag and a tobacco etch virus protease cleavage site. Protein expression in Escherichia coli BL21 (DE3) RIL to A600 nm = 0.6 was induced with 0.25 mm isopropyl β-d-galactoside in terrific broth at 16 °C for 16 h. Cells were collected by centrifugation at 4500 × g for 30 min, washed in PBS buffer, centrifuged again at 4500 × g for 15 min, and snap-frozen in liquid nitrogen. For protein purification cells were resuspended in lysis buffer (50 mm Tris-Cl (pH 8.0), 500 mm NaCl, 5 mm 2-mercaptoethanol), homogenized (Emulsiflex C-3, Avestin), and centrifuged at 7000 × g for 60 min. The supernatant was loaded onto a Ni2+ affinity column (HisTrap HP 5 ml, GE Healthcare), washed with 50 mm Tris (pH 8), 1 m NaCl, 5 mm 2-mercaptoethanol, and eluted in lysis buffer supplemented with 200 mm imidazole (pH 8.0). The His6 tag was cleaved with tobacco etch virus for 16 h at 4 °C during dialysis against lysis buffer. AtTTM3 was further purified by a second Ni2+ affinity step and by gel filtration on a Superdex 75 HR26/60 column (GE Healthcare) equilibrated in 25 mm Tris (pH 7.2), 250 mm NaCl, 5 mm 2-mercaptoethanol. Monomeric peak fractions were dialyzed against 20 mm Hepes (pH 7.4), 50 mm NaCl, 0.5 mm tris(2-carboxyethyl)phosphine and concentrated to 35 mg/ml for crystallization. Protein concentrations were estimated by protein absorption at 280 nm using the calculated molecular extinction coefficient. Site-specific mutations were introduced by PCR, and mutant proteins were purified like wild type.
The coding sequence of ygiF was amplified from genomic DNA (E. coli Mach 1 cells, Life Technologies) by PCR, and a synthetic gene coding for full-length mouse ThTPase (Uniprot ID Q8JZL3) and codon-optimized for expression in E. coli was obtained from Geneart (Life Technologies). Coding sequences were cloned into plasmid pMH-HT, and expression and purification were performed as described for AtTTM3.
Crystallization and Data Collection
Tetragonal AtTTM3 crystals (form A) developed at room temperature from hanging drops composed of 1.5 μl of protein solution and 1.5 μl of crystallization buffer (2.6 m NaCl, 0.1 m citric acid/NaOH (pH 5.0)) suspended over 1.0 ml of the latter as reservoir solution. Crystals were transferred in reservoir solution supplemented with 20% (v/v) ethylene glycol and 0.5 m KI for 20 s and snap-frozen in liquid nitrogen. Single-wavelength anomalous diffraction (SAD) data were collected on a Rigaku MicroMax rotating anode equipped with a copper filament, osmic mirrors, and an R-AXIS IV++ detector. Subsequently, an isomorphous native dataset was collected on a crystal originating from the same crystallization drop (see Table 1). A monoclinic crystal form (form B) developed in 20% (w/v) PEG 3350, 0.2 m NaCl, 0.1 m Bis-Tris (pH 7.0) and diffracted up to 1.3 Å resolution. Data processing and scaling were done with XDS (November 2014 version) (
). Hexagonal crystals for full-length ygiF grew at room temperature in hanging drops (1.5 μl and 1.5 μl) containing 0.2 m NaCl, 20 (w/v) PEG 3350. Crystals were transferred into crystallization buffer supplemented with 20% (v/v) ethylene glycol and snap-frozen in liquid nitrogen. For phasing, 0.1 m NaI was added to the cryo solution, and crystals were soaked for 5 min. SAD data were collected at a wavelength of 1.9 Å (see Table 2). Tetragonal mouse ThTPase crystals (form A) developed at room temperature from hanging drops composed of 1.5 μl of protein solution and 1.5 μl of crystallization buffer (27% (w/v) PEG 3350, 0.1 m Tris (pH 9.0), 0.2 m MgCl2) suspended over 1.0 ml of the latter as reservoir solution. Crystals were transferred into a reservoir solution supplemented with 20% (v/v) ethylene glycol and snap-frozen in liquid nitrogen. Monoclinic crystals (form B) developed in 1.6 m sodium/potassium phosphate buffer pH 6.8 (1:1 ratio) and were cryo-protected by stepwise transfer into a solution containing 1.6 m sodium/potassium phosphate buffer pH 6.8 and 20% (v/v) ethylene glycol.
TABLE 1Crystallographic data collection and refinement statistics for AtTTM3
AtTTM3-PPPi-Mg2+/Mn2+ form A crystals were transferred into the soaking solution (2.6 m NaCl, 0.1 Bis-Tris (pH 5), 10 mm sodium tripolyphosphate (Sigma), 10 mm MgCl2 (or MnCl2), 20% (v/v) ethylene glycol) by serial transfer to replace the citrate otherwise bound to the tunnel center.
For AtTTM3-PPPi-Mg2+/Mn2+, form B crystals were soaked for 20–30 min in 20% (w/v) PEG 3350, 0.2 m NaCl, 0.1 m Bis-Tris pH 7.0, 20% (v/v) ethylene glycol, 10 mm sodium tripolyphosphate, 5 mm MnCl2 using the same procedure as outlined above (substrate-bound structure).
For AtTTM3-Pi-Mn2+, AtTTM3 was co-crystallized with 5 mm sodium tripolyphosphate and 10 mm MnCl2 (product-bound structure).
For ygiF-PPPi-Mg2+/Mn2+, the substrate-bound complex was obtained by soaking (20–30 min) ygiF apo crystals in their crystallization buffer supplemented with 20% (v/v) glycerol, 10 mm sodium tripolyphosphate and 5 mm MgCl2 or MnCl2. ThTPase-ThTP-Mg2+. Form B crystals were soaked in crystallization buffer containing 20% (v/v) glycerol, 10 mm thiamine triphosphate and 10 mm MgCl2.
For ThTPase-ThDP-Mg2+, the product-bound structure was obtained by soaking form A crystals in a solution containing 10 mm sodium tripolyphosphate and 10 mm MnCl2 for 30 min. Two data sets were collected, one with λ = 1.0 Å and one close to the Mn-K edge (λ = 1.9 Å). No anomalous signal was found in the latter data set, possibly due to the high MgCl2 (0.2 m) concentration present in the crystallization buffer.
Structure Solution and Refinement
To solve the structure of AtTTM3, SAD and native data were scaled together with the program XPREP (Bruker AXS, Madison, WI) for SIRAS (single isomorphous replacement with anomalous scattering) phasing. The program SHELXD (
The structure of ygiF was solved by scaling redundant SAD data using XPREP. SHELXD located 15 consistent iodine and sulfur sites, which were input into SHARP for SAD site refinement and phasing at 2.7 Å resolution. Density modification and phase extension to 2.15 Å was carried out with PHENIX.RESOLVE (Table 2). The structure of mouse ThTPase was solved using the molecular replacement methods as implemented in the program PHASER and using the structure of the human ThTPase (PDB ID 3BHD) as a search model. Analysis with MolProbity (
) and purified by preparative high performance liquid chromatography.
NMR Time-course Experiment
A series of one-dimensional 31P NMR experiments was acquired at 310 K with a 600-MHz Bruker Avance-III spectrometer using a QXI probe-head allowing direct detection of 31P and equipped with a z-spoil gradient coil. 31P spectra were recorded using a relaxation delay of 1 s and an acquisition time of 42.6 ms (spectral width = 12,019.23 Hz). 128 scans were collected, resulting in a measurement time of 140 s per spectrum. 512 spectra were collected over a total acquisition time of ∼20 h. The enzymatic reaction was performed using 50 nm AtTTM3 and 5 mm sodium tripolyphosphate in 20 mm Bis-Tris propane (pH 8.5), 250 mm NaCl, 5 mm MgCl2 mixture. Deuterated water was added to a final concentration of 20% to the reaction mix before starting the experiment. Spectral parameters were calibrated and optimized on a 5 mm sodium tripolyphosphate sample to minimize time loss between the beginning of the reaction and the beginning of its observation by NMR. Spectra were processed using Topspin (version 2.1.6) (Bruker).
Phosphohydrolase Activities of AtTTM3 and ygiF Mutant Proteins
For the determination of the phosphohydrolase activity 2.5 nm AtTTM3 were incubated with 0.5 μm concentrations of the different phosphate-containing substrates in reaction buffer (150 mm NaCl, 20 mm Bis-Tris propane (pH 8.5), 5 mm MgCl2) at 37 °C. The reaction was stopped after 10 min, and the amount of free inorganic phosphate released was measured using the malachite green assay with minor modifications (
). 100 μl of reaction solution were mixed with 28 μl of dye mix (3 mm malachite green, 15% (v/v) sulfuric acid, 1.5% molybdate (w/v), 0.2% (v/v) Tween 20). After 5 min of incubation with the dye, the absorption at 595 nm was measured using a synergy H4 plate reader (Biotek). For each substrate a blank reaction was prepared lacking the enzyme. Controls either contained EDTA to a final concentration of 5 mm, or the enzyme was heat-inactivated at 95 °C for 5 min. To compare wild-type and mutant versions of AtTTM3 and ygiF, enzyme concentrations were increased to 180 nm to detect residual activity for some of the mutant proteins. Experiments were performed in triplicate; average values are plotted ±S.D.
Thermal shift assays were performed as previously described (
). Sypro Orange (Sigma) was added to wild-type and mutant AtTTM3 proteins in 25 mm Tris (pH 8.0), 250 mm NaCl, 5 mm 2-mercaptoethanol mixed with Sypro Orange to a final protein concentration of 10 μm. The protein-dye mixtures were loaded into a 96-well RT-PCR plate (Thermo Scientific), and measurements were performed using a C1000 thermal cycler (Bio-Rad). The fluorescence of SYPRO Orange was continously monitored at 570 nm, as a temperature gradient was applied (0.05 °C/s from 10 °C to 95 °C). Data were analyzed using the CFX Manager software (Bio-Rad). The maximum of the first derivative for each melting curve indicates the melting point of the protein. Experiments were performed in triplicate; average values are plotted ±S.D.
Polyphosphate Detection by UREA PAGE
Reactions contained 50 μm concentrations of the respective enzyme (AtTTM3, ygiF, mouse ThTPase, Vtc4p) in 150 mm NaCl, 20 mm Bis-Tris propane (pH 8.3), 1 mm MnCl2, and 10 mm ATP as substrate. Reactions were incubated at room temperature overnight. The reaction was stopped by adding Proteinase K (Sigma), and the resulting samples were mixed 1:1 with sample buffer (50% (w/v) urea, 2× Tris borate-EDTA, 20 mm EDTA (pH 8.0), 20% (v/v) glycerol, 0.25% (w/v) bromphenol blue). Samples were loaded onto a Tris borate-EDTA (TBE)-urea polyacrylamide gel (1× TBE, 7 m urea, 15% (v/v) polyacrylamide (19:1 acrylamide/bisacrylamide), 0.06% (w/v) tetramethylethylenediamine, 0.6% (w/v) ammonium persulfate) and stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI).
Results and Discussion
Our initial aim was to identify a polyP polymerase in plants using a combined structural and biochemical screen. We located three putative TTM proteins in Arabidopsis thaliana. AtTTM3 shares 12% sequence identity with Vtc4p (r.m.s.d. is 2.6 Å, comparing 149 corresponding Cα atoms and 1.1 Å comparing 77 Cα atoms in the tunnel center) and contains a conserved β-tunnel domain (
). We determined co-crystal structures of AtTTM3 with tripolyphosphate and Mg2+ or Mn2+ metal co-factors in two independent crystal lattices (Fig. 1). Crystal form B corresponds to the previously reported structure of AtTTM3 (PDB ID 3v85) in complex with a citric acid molecule (r.m.s.d is 0.3 Å comparing 203 corresponding Cα atoms) (
). We thus tested whether AtTTM3 can polymerize polyP from ATP or other nucleotide substrates but could not detect such an activity (Fig. 2B). Instead, AtTTM3 has specific, Mg2+ ion-dependent, short-chain polyphosphatase activity (Fig. 2C) as previously reported (
). We find that AtTTM3 hydrolyzes tripolyphosphate (PPPi) into pyro (PPi)- and orthophosphate (Pi) with a turnover rate of ∼10/s (Fig. 2, D and E). The Pi release from PPPi amounts to 27 ± 6/s when assayed by malachite green (see “Experimental Procedures”). AtTTM3 is not able to hydrolyze PPi but appears to catalyze the asymmetric cleavage of inorganic polyPn (n = 3–15), releasing Pi (Fig. 2, C and D).
Plant and Bacterial Tripolyphosphatases Contain Two Metal Coordination Sites
Comparison of AtTTM3 and Vtc4p revealed that four basic residues, which lead the growing polyP chain away from the active site in Vtc4p, are specifically replaced by four glutamate residues (Glu-2AtTTM3, Glu-4AtTTM3, Glu-90AtTTM3, and Glu171AtTTM3) in AtTTM3 (Fig. 3, A and E) (
). These residues, which form an acidic patch in the vicinity of the AtTTM3 tripolyphosphate substrate, are conserved among diverse TTM proteins with different catalytic activities (Figs. 3E and 4). The corresponding residues in the RNA triphosphatase Cet1p (Glu305Cet1p, Glu307Cet1p, and Glu-496Cet1p) coordinate a Mn2+ ion (
). As AtTTM3 efficiently hydrolyzes PPPi in the presence of Mg2+ ions only at neutral or basic pH, we used two crystal forms grown at pH 5.0 (form A) and pH 7.5 (form B) to crystallize substrate-metal complexes under non-hydrolyzable and hydrolyzable conditions, respectively. In both crystal forms, we determined structures of AtTTM3 in the presence of PPPi and either MgCl2 or MnCl2. In all our structures the catalytic Mg2+ ions can be substituted by Mn2+ ions, for which we calculated phased anomalous difference electron density maps from data collected near the Mn-K edge to confirm their modeled positions (TABLE 1, TABLE 2). At pH 5.0 (form A) we find a triphosphate moiety bound in the AtTTM3 active site. Here, PPPi coordinates a Mg2+/Mn2+ ion that acts as a bridge between the substrate and the invariant Glu-169AtTTM3 (Fig. 3A). The same metal co-factor coordination has been previously found in a Vtc4p AppNHp-Mn2+ complex (
) (Figs. 3A and 4A). A short soak at pH 7.0 (form B) shows the same arrangement of PPPi and Mn2+ in the active site (Fig. 1). However, when we co-crystallized AtTTM3 with its substrate at neutral pH, we found PPPi hydrolyzed and the tunnel center occupied by two orthophosphates and three Mn2+ ions (Fig. 3B). One of these Mn2+ ions is again found coordinated by the two phosphates (which correspond to the α and γ positions in PPPi; Fig. 4B) and by Glu-169AtTTM3, whereas the remaining metal coordination centers are formed by Glu-2AtTTM3, Glu-4AtTTM3, Glu-90AtTTM3, and Glu171AtTTM3 from the acidic patch conserved among many TTMs (Fig. 3, B and E).
To assess the functional relevance of the observed metal co-factors in AtTTM3, we studied substrate binding and metal ion coordination in the evolutionary distant TTM ygiF from E. coli. YgiF shares ∼18% sequence identity with AtTTM3 and has the same catalytic activity and substrate specificity (
). The structure of full-length ygiF was solved using iodide/sulfur SAD phasing and reveals the conserved TTM fold (r.m.s.d. with the AtTTM3 tunnel domain is 2.0 Å compared with 172 corresponding Cα atoms, and 0.65 Å comparing 75 corresponding Cα in the tunnel center) (Fig. 3C). ygiF contains a second, α-helical domain, which is connected to the TTM by a well ordered linker (residues 201–221, cyan in Fig. 3C) and which has been previously annotated as CHAD domain (
). The domain folds into two four-helix bundles of similar architecture and connectivity forming a V-shaped assembly (shown in red and orange in Fig. 3C). A structural homology search with the program DALI (
We next solved structures of ygiF in complex with PPPi and in the presence of either MgCl2 or MnCl2 (Table 2). The ygiF triphosphate substrate is bound in the same conformation as observed in AtTTM3, and we could identify two metal co-factors. One Mg2+/Mn2+ ion is again found coordinated by tripolyphosphate and by Glu-160ygiF, which corresponds to Glu-169 in AtTTM3 (position 1 in Fig. 3D). The second Mg2+/Mn2+ ion is coordinated by three glutamate residues from the acidic patch in ygiF (Glu-4ygiF, Glu-6ygiF, Glu-162ygiF) and by a water molecule that is positioned close to a terminal phosphate of the tripolyphosphate substrate (position 2, Fig. 3, D and E). When we calculated phased anomalous difference maps from diffraction data collected near the Mn-K edge, we found that the ygiF metal binding site 1 shows a higher occupancy compared with the second site (peak heights are 125 and 45 σ, respectively), whereas we cannot detect difference density at the third metal position found in AtTTM3 (Fig. 3D). Taken together, our experiments define two consistent Mg2+/Mn2+ coordination centers in plant and bacterial TTM tripolyphosphatases.
We compared our structures to other TTM enzymes for which metal ion-bound complexes have been reported. In the RNA triphosphatase Cet1p, a Mn2+ ion is bound by the acidic patch, and its position corresponds to site 2 in ygiF and AtTTM3 (Figs. 4C and 3E) (
). Here, the octahedral coordination of the Mg2+/Mn2+ ion is completed by an glutamate residue (Glu-169AtTTM3, Glu-160ygiF), which is invariant in all TTM proteins characterized thus far (Figs. 3D and 4). A second binding site is formed by three glutamate residues originating from an acidic patch conserved among many but not all TTM enzymes (Figs. 3, A, D, and E, and 4) (
). This metal ion is positioned close to a terminal phosphate in our AtTTM3 and ygiF substrate complex structures (Fig. 3D) and may thus be involved in catalysis rather than in substrate binding.
Structures of Mammalian Thiamine Triphosphatase Reveal the Location of the γ-Phosphate
We investigated the contributions of the two metal ion centers to TTM substrate binding and catalysis. A conceptual problem with the analysis of our plant and bacterial tripolyphosphatases is that they catalyze the asymmetric cleavage of a symmetric substrate (Fig. 2) (
). It is thus difficult to assess in crystal structures, which terminal phosphate represents the γ-phosphate that is being hydrolyzed (Fig. 3, A and B). We thus structurally characterized a mammalian TTM ThTPase, which was previously shown to specifically hydrolyze thiamine triphosphate (ThTP) into ThDP and Pi (
). Consequently, we found an intact ThTP molecule bound in the tunnel center of ThTPase (Fig. 5A). The thiamine portion of the substrate binds to a pocket generated by the tunnel walls and the C-terminal plug helix, with the thiazole ring making a stacking interaction with Trp-53 and with Met-195 from the plug helix (Fig. 5A). The ThTP triphosphate moiety binds in the same conformation as outlined for the PPPi-bound structures of AtTTM3 and ygiF above. Our substrate-bound mouse ThTPase structure supports an earlier docking model of human ThTPase (
). Indeed, we found a product complex trapped in the active site of the enzyme, with a ThDP molecule and an orthophosphate located in the tunnel center (Fig. 5A). ThDP is coordinated by Arg-55 and Arg-57 in the substrate binding site but no longer allows for the coordination of a Mg2+/Mn2+ ion in metal binding site 1, possibly because the missing γ-phosphate would be required for Mg2+/Mn2+ coordination (Fig. 5A). The γ-phosphate in our structure apparently has been hydrolyzed, and the resulting Pi has slightly moved away from the tunnel center (Fig. 5A). It is now found coordinated by Arg-125 and in direct contact with a Mn2+ ion located in metal binding site 2, reinforcing the notion that this metal ion may play a crucial role in catalysis (Fig. 5A).
TTM Proteins Use a Two-metal Catalytic Mechanism
The structural features surrounding metal binding site 2 in our structures allow proposing a unified catalytic mechanism for triphosphate tunnel metalloenzymes. In our PPPi-bound ygiF structure we find a water molecule coordinated by the second Mn2+ ion, which is in an ideal position to serve as the activated nucleophile (Fig. 5, B and C). Indeed, structural superposition with a bacterial adenylate cyclase (
) reveals that its cAMP O3′ group, which acts as the nucleophile in the cyclization reaction, is located in the same position as the water molecule in our ygiF structure (Fig. 5B). Consistently, this position also is occupied by an oxygen atom of a product orthophosphate, which we located in our AtTTM3 post-catalysis structure (see above, Figs. 3B and 5B). Based on these findings, we propose that metal binding site 1 is required for proper substrate coordination in TTM proteins (
), and metal ion 2 activates a water molecule to allow for a nucleophilic attack on the triphosphate substrate (Fig. 5C). This would rationalize why the glutamate residues from the acidic patch, which are involved in the coordination of the second metal ion, are well conserved among TTM proteins (Figs. 3E and 4). The basic residues in the tunnel center appear to be mainly involved in substrate coordination (Figs. 2A and 3A); however, the invariant Arg-56ygiF (Arg-52AtTTM3) possibly activates the substrate for the nucleophilic attack by forming a hydrogen bond with the oxygen atom connecting the β- and γ-phosphate of the substrate (Fig. 5, B and C). The suggested TTM reaction mechanism is reminiscent of the one found in mammalian type V adenylate cyclases (
We next performed a mutational analysis of substrate- and metal co-factor-interacting residues in AtTTM3 and ygiF (Fig. 6). Mutation of Glu-2AtTTM3, Glu-4AtTTM3, and Glu-169AtTTM3 to Asp or Ala strongly reduces the PPPi hydrolysis of the plant enzyme (Fig. 6, A and B; see Fig. 7 for mutant protein stability). Consistently, changing the corresponding Glu-6ygiF and Glu-160ygiF to Ala impairs the enzymatic activity of ygiF, suggesting that the proper arrangement of metal binding sites 1 and 2 in the tunnel center is essential for catalysis (Fig. 6, A–C). Mutation of Arg-52AtTTM3 or Arg-56ygiF, but not of the neighboring Arg-54AtTTM3, to Ala again strongly inhibits catalysis, highlighting its potential role as proton donor (Fig. 6, A–C, see above). In addition, we find moderately reduced enzymatic activities upon the mutation of Lys-76AtTTM3 to Leu or Ala (Fig. 6, A and B). This residue and the corresponding Lys-69ygiF appear to be involved in substrate binding and orient Glu-2AtTTM3/Glu-4ygiF to coordinate metal ion 2 (Fig. 6, A and B). Glu-2AtTTM3 is also in contact with Glu-90AtTTM3, mutation of which to Ala again reduces the enzymatic activity of the Arabidopsis enzyme (Fig. 6, A and B). Taken together, our and previous mutational analyses (
). The basic residues, with the exception of the catalytic Arg-52AtTTM3 appear to be mainly involved in substrate coordination in the tunnel center (Fig. 6, A–C). Further experimentation will be required to rationalize the specific effects of certain point mutations on substrate/metal co-factor binding or catalysis itself.
Comparison with Vtc4p suggests that the first but not the second metal binding site are present in the yeast polyphosphate polymerase (Figs. 2, A and B, and 4A) (
). Importantly, mutation of Lys-200AtTTM3, which corresponds to the catalytic Lys-458Vtc4p, to either Leu or Ala has little effect on tripolyphosphatase activity of the plant enzyme (Fig. 6, A and B) (
). This suggests that there are significant mechanistic differences between TTM polyphoshatases and polyphosphate polymerases despite their strong structural homology (Figs. 2, A and B, and 3A).
Directionality of Substrate Binding Defines TTM Catalytic Activity
To better understand how acidic-patch containing TTMs are able to carry out very different enzyme reactions, we superimposed our substrate-bound structures of AtTTM3, ygiF, and mouse ThTPase with an ATP-analog complex of a bacterial TTM adenylate cyclase (
) (Fig. 8). We found that although the triphosphate parts of all ligands closely align in the tunnel center, their “tail” moieties can enter the tunnel domain from opposite sites in different enzymes (Fig. 8). The unique modes of substrate binding in ThTPases and adenylate cyclases allow these enzymes to carry out rather different reactions and to produce different leaving groups (ortho- and pyrophosphate, respectively) while maintaining a unified cleavage site in close proximity of metal binding site 2. Both the N- and C-terminal sides of the tunnel domain have evolved to recognize specific substrates, as exemplified by our ThTPase structure and by the class IV adenylate cyclase complexes (Figs. 5, A and B, and 8) (
). The observed substrate binding mode in mouse ThTPase reinforces the notion that to bind their substrates, TTMs require opening of their closed tunnel domains into a cup-shaped hand, as previously shown by NMR spectroscopy (
Members of the ancient triphosphate tunnel metalloenzyme family can be found in all kingdoms of life. Our comparative analysis defines that most of these enzymes share a common catalytic mechanism in their tunnel centers, yet they have evolved different substrate recognition modes on their tunnel entries. The observed substrate plasticity apparently allows TTM proteins to act on a wide array of enzyme substrates and to perform very different reactions, many of which likely remain to be discovered.
J. M. and M. H. designed the study and wrote the paper. V. T. performed NMR titrations, J. M. performed all other experiments. All authors analyzed data and approved the final version of the manuscript.
We thank T. Baiga for help with ThTP synthesis, members of the Hothorn laboratory for discussion, and W. Saenger for suggestions and critical reading of the manuscript. Diffraction experiments were performed on the PXII and PXIII beam lines at the Swiss Light Source, Paul Scherrer Institut, Villigen, Switzerland and at beam line ID29 of the European Synchrotron Radiation Facility (ESRF), Grenoble, France.
Inorganic polyphosphate: essential for growth and survival.