Article pubs.acs.org/IC
Phosphate Hydrolysis by the Fe2−Ca3‑Dependent Alkaline Phosphatase PhoX: Mechanistic Insights from DFT calculations Rong-Zhen Liao*,† and Per E. M. Siegbahn‡ †
Key Laboratory of Material Chemistry for Energy Conversion and Storage, Ministry of Education, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China ‡ Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, SE-10691 Stockholm, Sweden S Supporting Information *
ABSTRACT: PhoX is a pentanuclear metalloenzyme that employs two ferric ions and three calcium ions to catalyze the hydrolysis of phosphomonoesters. On the basis of the X-ray structure of PhoX (Science 2014, 345, 1170−1173), a model of the active site is designed, and quantum chemical calculations are used to investigate the reaction mechanism of this enzyme. The calculations support the experimental suggestion, in which the two high spin ferric ions interact in an antiferromagnetic fashion. The two step mechanism proposed by experimentalists has been investigated. The nucleophilic attack of a trinuclear bridging oxo group on the phosphorus center was calculated to be the first step, which is concomitant with the departure of the phenolate, which is stabilized by a calcium ion. The second step is a reverse attack by a water molecule activated by a calciumbound hydroxide, leading to the regeneration of the bridging oxo group. The second step was calculated to have a barrier of 27.6 kcal/mol. The high barrier suggests that the alternative mechanism involving phosphate release directly from the active site seems to be more likely. All five metal ions are involved in the catalysis by stabilizing the pentacoordinated trigonal bipyramidal transition states.
1. INTRODUCTION Alkaline phosphatases are a class of enzymes that catalyze phosphoryl transfer from a broad range of monoesters.1−4 When the environmental phosphate concentration is low, microorganisms can use extracytoplasmic alkaline phosphatases to obtain phosphate from nutrient. Two prominent families, namely, PhoA5−7 and PhoX,2,8−10 have been found to be widely distributed in bacteria, archaea, and eukarya. PhoA is a trinuclear metalloenzyme incorporating two Zn ions and one Mg ion in the active site,11 and its reaction mechanism has been studied extensively by both experimental12−17 and computational tools.18−22 Many fewer efforts were devoted to the study of PhoX, and amino acid sequence comparisons showed that PhoX has no sequence similarity to other phosphoryl transfer enzymes, including PhoA.8 On the basis of metal reconstitution experiments, PhoX had been considered to be an exclusively Ca-dependent enzyme.10,23 Very recently, crystal structure analysis showed that PhoX from Pseudomonas f luorescens embraces a unique complex pentanuclear active site (Figure 1), including two iron ions (Fe1 and Fe2), three calcium ions (Ca1, Ca2, and Ca3), and an oxo species (O1) bridging the two iron ions and one calcium ion.24 Both iron ions have been determined to be in the high spin ferric form interacting in an antiferromagnetic fashion based on electron paramagnetic resonance (EPR) spectroscopy studies.24 Fe1, Fe2, and Ca1 form almost an equilateral triangle, with sides of about 3.6 Å. These three ions are bridged by Glu194, Glu273, phosphate, a central oxo O1, and a second oxygen species, presumably a water molecule. In addition, two © 2015 American Chemical Society
Figure 1. Crystal structure of the active site of the wild-type PhoX from Pseudomonas f luorescens complexed with phosphate (coordinates extracted from PDB code 4ALF).24 The five metal ions and the bridging oxo (O1) are labeled in blue.
aspartates (Asp292 and Asp479), two glutamates (Glu90 and Glu387), a cysteine (Cys179), and three water molecules are Received: October 3, 2015 Published: December 10, 2015 11941
DOI: 10.1021/acs.inorgchem.5b02268 Inorg. Chem. 2015, 54, 11941−11947
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Inorganic Chemistry
Scheme 1. Proposed Phosphoryl Transfer Mechanism24 with the Alternative Direct Phosphate Release Also Shown for Comparison
number of enzymes, including a few phosphoryl transfer metalloenzymes.28−36
ligated to these three metal ions, as shown in Figure 1. Furthermore, both Ca2 and Ca3 are involved in phosphate binding, and they are bridged by Asp494. Ca2, with two water molecules bound, is further bridged to Fe1 via Glu387, and to Ca1 via Asp479. Ca3 has a nonbridging negatively charged residue Glu532 and three water molecules coordinated. Several important second-shell residues, Asp69, Arg385, Thr534, Gln550, are hydrogen-bonded to some of the first-shell ligands. On the basis of crystallographic and kinetic studies as well as the mechanisms of the related phosphoryl transfer enzymes, the following mechanistic scenario has been put forward for PhoX (Scheme 1).24 After substrate binding, the bridging oxo (O1) performs an inline nucleophilic attachment on the phosphorus moiety, which is coupled with the departure of the leaving alkoxide or phenolate group, stabilized by the Ca3 ion. Subsequently, a water molecule from solution enters and delivers a proton to the negatively charged leaving group, leading to a Ca3-bound hydroxide. Next, this Ca3-bound hydroxide performs a reverse inline attack on the phosphorus moiety to regenerate the trinuclear bridging oxo. An alternative mechanism for the regeneration of the bridging oxo is a direct phosphate release from the active site (Scheme 1). This mechanism has been suggested to be less likely due to the quite strong binding between the phosphate and the pentanuclear cluster.24 This enzyme uses a trinuclear bound oxo group as the nucleophile, and differs from many other phosphatases, which use either a dinuclear bridging hydroxide25−35 or a mononuclear bound Ser alkoxide.18−22,36 Here, density functional theory (DFT) has been used to elucidate the mechanism of the phosphomonoester hydrolysis utilized by PhoX. With a model of the active site first designed on the basis of X-ray structure (PDB code 4ALF), the hybrid functional B3LYP*-D3 (B3LYP37 functional with 15% HF exchange38 and D3 dispersion39 from B3LYP) is then used to compute the energy profile for the hydrolysis of a representative substrate, p-nitrophenyl phosphate. This kind of approach has already been successfully used to study a large
2. COMPUTATIONAL DETAILS 2.1. Active Site Model. A model of the PhoX active site was built on the basis of the X-ray structure of the enzyme phosphate complex (PDB ID: 4ALF).24 The model (Figure 2) consists of the two ferric ions, the three calcium ions, along with their first-shell ligands Glu90, Cys179, Glu194, Glu273, Asp292, Glu387, Asp479, Asp494, Glu532, bridging oxo, and eight water molecules. Furthermore, four essential second-shell residues, Asp69, Arg385, Thr534, and Gln550, were also added in the model. Hydrogen atoms were added manually, and all amino acids were truncated at their α-carbon atoms and saturated with hydrogen atoms. Thus, only the side chains of the residues were kept
Figure 2. Optimized structure of the model of PhoX. Atoms labeled with red circle were frozen during the geometry optimizations. 11942
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Inorganic Chemistry in the model. The α-carbon and one hydrogen atom of each residue were kept frozen to their X-ray structure positions during geometry optimizations to mimic the steric effects imposed by the protein environment. The p-nitrophenyl phosphate substrate, which has experimentally been used for activity measurement,24 was chosen as a representative for the enzymatic hydrolysis of phosphate monoesters. The model is composed of 210 atoms and has a total charge of −1. 2.2. DFT Calculations. The calculations presented herein were carried out using the Gaussian0940 code with the B3LYP37 hybrid functional. Geometry optimizations were performed using the LANL2DZ41 pseudopotential for Fe, 6-311+G(d) for P, and 631G(d,p) for S, Ca, C, N, O, H (labeled as BSI). Both the antiferromagnetically coupled singlet (high spin on each iron) and the ferromagnetically coupled high spin 11-tet were considered for all calculations. To obtain more accurate energies, single-point calculations were performed on the optimized structures using the B3LYP*38 functional and the larger basis sets 6-311+G(2df,2p) for all elements except Fe, for which the LANL2TZ(f)42 basis sets (with pseudopotential) were used (labeled as BSII). B3LYP* has been shown to give better relative energies of different spin states in transition metal complexes.38 Grimme’s D3 dispersion corrections (with the default D3 damping function)39 were added at single-points. To evaluate the solvation effect of the protein environment that is not explicitly considered in the active site model, the SMD43 solvation model method was applied for single-point calculations at the B3LYP/ BSI level. The dielectric constant was set to be 4, which is commonly used in the quantum chemical cluster approach.44 The NBO5.G45 as embedded in the Gaussian09 program was used to calculate the Wiberg bond indices (WBI)46 on the optimized structures at the B3LYP/BSI level. Analytical frequency calculations were carried out to estimate vibrational zero-point energies (ZPE). As shown below, certain atoms at the periphery of the model were kept frozen to their X-ray positions. This technique introduces several small imaginary frequencies, usually on the order of 10−50i cm−1, which contribute little to the ZPE and can therefore be disregarded. 2.3. Broken-Symmetry Calculation and Noodleman Correction. Experimentally, the enzyme is known to be two high spin ferric ions interacting in an antiferromagnetic fashion (SA = 5/2, SB = 5/2, Stotal = 0).24 The antiferromagnetically coupled state was obtained using the broken-symmetry (BS) approach.47−51 The spin Hamiltonian H for homovalent magnetic dimers can be written via the Heisenberg coupling constant J: H = − 2JSA ·SB
(1)
The energy difference between ferromagnetically coupled high spin 11-tet (Stotal = Smax = SA+SB = 5) and BS (Stotal = Smin = SA − SB = 0) low spin singlet states can be described by
EHS − EBS = − 4JSA ·SB = − 25J
(2)
, in which the high spin energy EHS was calculated from a single-point calculation on the optimized BS structure (with energy EBS). Then, the pure-spin ground state energy E0 (S = 0) according to the BS geometry can be estimated by
E0 = E HS + JSmax(Smax + 1) − JSmin(Smin + 1) = E HS + 30J (3)
3. RESULTS AND DISCUSSION The structure of the PhoX active site model with p-nitrophenyl phosphate bound (React) was first optimized. The full model is displayed in Figure 2 while the core part of the full model is in Figure 3. React is a broken-symmetry singlet with the two high spin ferric ions (S = 5/2) interacting in an antiferromagnetic fashion. On Fe1, the spin density is 4.09, while it is −3.98 on Fe2. The Heisenberg coupling constant J was calculated to be −0.257 kcal/mol, which can be converted into −89.9 cm−1. The antiferromagnetic coupling of intermediate (3α3β) and
Figure 3. Optimized structures for the hydrolysis of p-nitrophenyl phosphate. Distances are given in ångstroms, and Mulliken spin densities are shown in red italic type. For clarity, only the core of the model is shown (Figure 2 for full model).
low (1α1β) spin states has also been considered. However, their energies were calculated to be 35.4 and 49.8 kcal/mol (Table 1), respectively, which are too high to be accessible. In addition, the closed-shell singlet lies at 75.7 kcal/mol. The 11943
DOI: 10.1021/acs.inorgchem.5b02268 Inorg. Chem. 2015, 54, 11941−11947
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kinase.56 TS1 has an imaginary frequency of 246.1i cm−1, which corresponds to the P−O1 bond formation and the P− O3 bond cleavage. For the singlet state, the barrier (Figure 4)
Table 1. Relative Energies (kcal/mol), Fe−Fe Distances (Angstrom), and Spin Densities on Fe for Different Spin States of Reacta
singlet(5α5β) singlet(3α3β) singlet(1α1β) singlet(0α0β) 11-tet nonet septet quintet triplet
relative energy
Fe−Fe distance
spin on Fe1
spin on Fe2
0 35.4 49.8 75.7 7.1 16.4 25.4 43.7 50.0
3.25 3.25 3.21 3.15 3.29 3.31 3.27 3.26 3.21
4.09 2.78 0.93 0 4.11 4.06 4.08 0.88 0.93
−3.98 −3.41 −1.12 0 4.00 3.18 1.16 3.17 1.13
Note: “stable = opt” has been used to ensure that all optimized wave functions are stable.
a
energy of the ferromagnetically coupled high spin 11-tet is 7.1 kcal/mol higher than the singlet state. Other spin states, including triplet, quintet, septet, and nonet, are all found to have much higher energy. These results agree quite well with the experimental data from EPR studies, which suggest two high spin ferric ions interacting in an antiferromagnetic fashion.24 In React, the Fe1−Fe2 distance in the singlet state was calculated to be 3.25 Å, which is slightly shorter than that in the high spin 11-tet (3.29 Å) due to the strong antiferromagnetic coupling between the two ferric ions. This distance is also quite close to those in the X-ray structures, which are in the range 3.36−3.59 Å depending on the ligands bound to the active site.24 Both ferric ions are hexacoordinated while all three calcium ions are heptacoordinated. The unique feature of this enzyme, the structure of the trinuclear bridging oxo, is wellreproduced, and the Fe1−O1, Fe2−O1, and Ca1−O1 distances are 1.82, 1.96, and 2.46 Å, respectively. The NBO charge on this nucleophilic bridging oxo is −0.90, while it is +2.51 on the electrophilic phosphorus center. Both Fe1 and Fe2 are involved in phosphate substrate binding via coordination to two of the phosphate oxygen atoms. This kind of substrate binding mode has also been seen in a number of multinuclear zinc enzymes,28,30−32 in which two zinc ions bind two of the phosphate oxygen atoms. By doing so, the substrate is welloriented and ready for the following nucleophilic attack. Importantly, PhoX employs two additional calcium ions (Ca2 and Ca3) to bind the third oxygen atom of the phosphate substrate, and the Ca2−O4 and Ca3−O4 distances are 2.45 and 2.42 Å, respectively. Four metal ions are thus involved in the phosphate substrate binding, which is quite unusual in phosphatases. The improper phosphate dihedral angle of O6−P−O4−O5 is approximately 120° in solution, while it becomes 140.5° in React when the substrate is bound. This implies a more accessible conformation of the phosphate after its coordination, which can assist the following nucleophilic attack. Furthermore, the P−O1 distance is 2.88 Å, and the O1− P−O3 angle is 165.6°, implying a nearly perfect in-line attack of the trinuclear oxo on the phosphorus moiety. We find that the reaction proceeds via a concerted SN2-type transition state TS1 (Figure 3), and no pentacoordinated intermediate can be located. This is similar to the results of RNase Z,30 phospholipase C,31 Nuclease P1,32 endonuclease IV,52 myosin,53 human uridine-cytidine kinase 2,54 cyclindependent kinase Cdk2,55 and cAMP-dependent protein
Figure 4. Calculated potential energy profile for the hydrolysis of pnitrophenyl phosphate.
was calculated to be 11.8 kcal/mol (14.6 kcal/mol in the 11tet), and the resulting intermediate Int1 was found to be 6.5 kcal/mol (5.8 kcal/mol in the 11-tet) lower than React. For the high spin 11-tet, the barrier is 7.5 kcal/mol (14.6 kcal/mol relative to Reactsinglet), and the energy of Int1 is −12.9 kcal/ mol (−5.8 kcal/mol relative to Reactsinglet). At TS1, the scissile P−O3 bond is 1.92 Å, and the nascent P−O1 bond is 2.10 Å. A More O’Ferral Jencks (MFJ) plot57,58 (Figure 5) suggests that
Figure 5. More O’Ferral Jencks plot for the hydrolysis of pnitrophenyl phosphate.
TS1 is quite synchronous. The angle of O1−P−O3 is 174.6°, which is consistent with an in-line attack. All five metal ions help stabilize the pentacoordinated trigonal bipyramidal transition state. In addition, a Ca2-bound water molecule is hydrogen-bonded to the leaving oxygen, with a H1−O3 distance of 1.80 Å. Downhill from TS1, the leaving p-nitrophenolate product becomes coordinated to Ca3 (structure labeled as Int1), and is further stabilized via a hydrogen bond to the Ca2-bound water molecule. A high degree of structural consistency can be observed from the overlaid structure of React, TS1, and Int1 (see Figure S1 in the Supporting Information), and the major 11944
DOI: 10.1021/acs.inorgchem.5b02268 Inorg. Chem. 2015, 54, 11941−11947
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Inorganic Chemistry geometric change happens in the reacting region. The pnitrophenolate product can easily escape from the enzyme active site upon abstracting a proton from the Ca2-bound water molecule. However, the direct release of the phosphate from Int1 was suggested to be energy demanding as it is strongly coordinated to the pentanuclear center.24 Indeed, it was proposed that a water molecule from solution performs the reverse attack on the phosphorus moiety (Scheme 1). Consequently, the bridging oxo can be regenerated to start the next catalytic cycle.24 Therefore, the phenol product was manually replaced by a water molecule, and the geometry (called Int2, Figure 6) was optimized. The energetic requirement for the conversion of Int1 to Int2 can be roughly obtained using the following equation: Int1(in enzyme) + H 2O(in water solution) → Int2(in enzyme) + p‐nitrophenol(in water solution)
It should be pointed out that Int1 and Int2 represent models in the enzyme, for which the solvation effects from the protein surrounding were calculated using a dielectric constant of 4, and the solvation effects for water and p-nitrophenol in water solution were calculated using a dielectric constant of 78.5. The use of different dielectric constant thus represents the different environment for different species (Int1 and Int2 in enzyme, vs water and p-nitrophenol in water). Accordingly, this step was calculated to be endothermic by 12.3 kcal/mol. It should be pointed out that the solvation energies of the water molecule and the phenol product may not be quite accurate, and also the entropy effect is not included here. Therefore, the uncertainty for this step could be several kcal/mol. The ground state of Int2 was calculated to be 11-tet, and the low spin singlet is 1.0 kcal/mol higher in energy. Both Int1 and Int2 have much weaker spin coupling between the two ferric ions as compared with React. The reason for this is that the Fe−Fe distances are around 3.8 Å at Int1 and Int2, which is much longer than that at React (3.25 Å). At Int2, a Ca2-bound hydroxide is formed, and this hydroxide is further stabilized by Arg385 and the incoming water molecule via two hydrogen bonds. Any attempt to optimize a structure with a Ca3-bound hydroxide, similar to the one proposed in Scheme 1, leads to a barrierless proton transfer from the Ca2-bound water molecule to this Ca3-bound hydroxide. One major reason is that the Ca2-bound hydroxide can be stabilized via hydrogen bond to Arg385. This is different from the experimental suggestion (Scheme 1), in which a Ca3-bound hydroxide was proposed to be a key intermediate.24 This Ca2-bound hydroxide can then function as a general base to abstract a proton from the incoming water molecule to generate a hydroxide for the reverse attack. From Int2, the transition state for the reverse water attack (TS2, Figure 6) was optimized and is shown in Figure 6. The barrier was calculated to be 15.3 kcal/mol compared with Int2. The key geometry of TS1 and TS2 was found to be quite similar. The P−O1 and P−O3 distances are 1.95 and 1.88 Å. MFJ analysis (Figure 5) shows that TS2 has a more associative character than TS1. This reverse attack leads to the generation of Prod with the regeneration of the trinuclear bridging oxo group. This step (from Int2 to Prod) is exothermic by 5.2 kcal/ mol in the singlet state. The calculated energy profile for p-nitrophenyl phosphate hydrolysis is displayed in Figure 4. The second step is ratelimiting, with a barrier of 27.6 kcal/mol (TS2 relative to Int1).
Figure 6. Optimized structures of stationary points for the reverse water attack. Distances are given in ångstroms, and Mulliken spin densities are shown in red italic type.
Single-point calculations using M06-D359 have also been carried out, and it gave a total barrier of 29.0 kcal/mol, quite close to the one obtained from B3LYP*-D3. Since experimental kinetic data has not been reported, a direct comparison of the barrier from calculation and kcat from experiment is not 11945
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Inorganic Chemistry possible. Considering that a typical kcat of an enzyme is in the order of s−1, which can be translated into barrier of around 18 kcal/mol, the calculated barrier seems to be overestimated. The main reason could come from the overestimation of the ligand exchange energy (from Int1 to Int2). The high barrier for the reverse attack suggests that this mechanism seems to be quite unlikely, and the direct release of the phosphate product from the enzyme active site seems to be more likely. It should be pointed out that the cluster approach used cannot model the phosphate release from the active site properly. The main reason is that the product release is a very complex process involving the movement of the protein surrounding, proton transfer, and the binding of water molecules. The alternative QM/MM molecular dynamics approach may be used to understand this process in the future. In addition, further experimental studies might be helpful to distinguish these two scenarios.
Foundation. Computer time was generously provided by the Swedish National Infrastructure for Computing.
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4. CONCLUSION In this paper, density functional calculations have been utilized to investigate the mechanism of phosphomonoester hydrolysis by PhoX. The calculations establish that the two ferric ions in the active site are both in their high spin states interacting in an antiferromagnetic fashion, forming a broken-symmetry singlet state. The phosphate substrate binds to the pentanuclear active site mainly via direct coordination to Fe1, Fe2, Ca2, and Ca3. The trinuclear bridging oxo group performs an in-line nucleophilic attack on the phosphorus center, in association with the departure of the phenolate product. No protonation of the phenolate is needed as Ca3 can stabilize it via coordination. During the attack, the Fe1−Fe2 distance increases, and consequently, the spin coupling between the two ferric ions becomes weaker. Next, the protonation of the phenolate by a Ca2-bound water molecule takes place, followed by the displacement of the phenol product by a water molecule. In the following step, the incoming water molecule delivers a proton to the Ca2-bound hydroxide and makes the reverse attack leading to the reformation of the trinuclear bridging oxo group. All five metal ions are involved in lowering the barrier by stabilizing the pentacoordinated trigonal bipyramidal transition states. The quite high barrier for the second step (27.6 kcal/ mol) suggests that phosphate release directly from the active site seems to be more likely.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.5b02268. Overlaid structures and Cartesian coordinates (PDF)
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21503083), startup funding from Huazhong University of Science and Technology, the Swedish Research Council, and the Knut and Alice Wallenberg 11946
DOI: 10.1021/acs.inorgchem.5b02268 Inorg. Chem. 2015, 54, 11941−11947
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DOI: 10.1021/acs.inorgchem.5b02268 Inorg. Chem. 2015, 54, 11941−11947
