Molecular Mechanism of the Reaction Specificity in Threonine Synthase

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Molecular Mechanism of the Reaction Specificity in Threonine Synthase: Importance of the Substrate Conformations Yuzuru Ujiie, Wataru Tanaka, Kyohei Hanaoka, Ryuhei Harada, Megumi Kayanuma, Mitsuo Shoji, Takeshi Murakawa, Toyokazu Ishida, Yasuteru Shigeta, and Hideyuki Hayashi J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 10 May 2017 Downloaded from http://pubs.acs.org on May 14, 2017

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The Journal of Physical Chemistry

Molecular Mechanism of the Reaction Specificity in Threonine Synthase: Importance of the Substrate Conformations

Yuzuru Ujiie,a Wataru Tanaka,a Kyohei Hanaoka,a Ryuhei Harada,b Megumi Kayanuma,b Mitsuo Shoji,*,a,b Takeshi Murakawa,c Toyokazu Ishida,d Yasuteru Shigeta,a,b and Hideyuki Hayashi e

a

Graduate School of Pure and Applied Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba,

Ibaraki 305-8571, Japan b

Center for Computational Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba 305-8577,

Japan c

Department of Biochemistry, Osaka Medical College, Takatsuki 569-8686, Japan.

d

Nanosystem Research Institute, National Institute of Advanced Industrial Science and Technology

(AIST), Tsukuba, Ibaraki 305-8568, Japan e

Department of Chemistry, Osaka Medical College, Takatsuki 569-8686, Japan.

* Corresponding authors at: Center for Computational Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8577, Japan (M. Shoji) E-mail addresses: [email protected] (M. Shoji).

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Abstract Threonine Synthase (ThrS) catalyzes the final chemical reaction of L-threonine biosynthesis from its precursor, O-phospho-L-homoserine. As the phosphate ion generated in its former half reaction assists its latter reaction, ThrS is recognized as one of the best examples of product-assisted catalysis. In our previous QM/MM study, the chemical reactions for the latter half reactions, which are critical for the product-assisted catalysis, were revealed. However, accurate free energy changes caused by the conformational ensembles and entrance of water molecules into the active site are unknown. In the present study, by performing long-time scale MD simulations, the free energy changes by the divalent anions (phosphate or sulfate ions) and conformational states of the intermediate states were theoretically investigated. We found that the calculated free energy double differences are in good agreement with the experimental results. We also revealed that the phosphate ion contributes to forming hydrogen bonds that are suitable for the main reaction progress. This means that the conformation of the active site amino acid residues and the substrate, and hence, the tunable catalysis, are controlled by the product phosphate ion, and this clearly demonstrates a molecular mechanism of the product-assisted catalysis in ThrS.


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1. Introduction Threonine synthase (ThrS) catalyzes the formation of L-threonine from O-phospho- L-homoserine (OPHS) in the final step of the threonine biosynthetic pathway.1,2 ThrS possesses pyridoxal-5′ -phosphate (PLP) in the active site (see Figure 1(a)),3-5 and belongs to a family of PLP-dependent enzymes that catalyze manifold reactions in the metabolism of amino acids.6-10 ThrS processes the most complicated reaction of the PLP enzymes, and all seven types of the intermediate states known for PLP enzymes are formed during its catalytic cycle. As the result, there are many chances of side reactions taking place, but ThrS carries out only one specific, well organized reaction. Therefore, knowing the mechanism by which ThrS controls strictly the versatile catalyst PLP and attains its reaction specificity has an extraordinary importance not only in pure enzymology but also in biotechnology for the semisynthetic production of medical and industrial compounds.9 ThrS is also attractive to be a potential target for developing novel antibiotics, antifungal agents and herbicides, because the threonine biosynthetic pathway is carried out only in bacteria, yeast, and plants, and not in mammals.11,12 The reaction steps of ThrS have been considered as follows: the initial PLP–Lys aldimine state (1) reacts with OPHS, and after at least seven intermediate states (2−8), the initial state 1 is regenerated (full catalytic cycle is shown in Figure S1, Supporting Information).5 The reaction cycle can be divided into two parts. In the first-half reaction (1 →→ 6), the γ-phosphate group of OPHS is eliminated, and in the second-half reaction (6 → 7 → 8 → 1), the addition of water to 6 produces L-threonine. The intermediate state 6 is a PLP‒α-aminocrotonate aldimine state, which possesses a characteristic large absorption band around 450 nm, and locates at the branching point of the two reaction pathways. In the main reaction (6 → 7 → 8 → 1), 6 is attacked by a water molecule with the aid of a general base catalysis by the ε-amino group of Lys61 to give the




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carbanion intermediate state 7, which is then protonated to form PLP–L-threonine aldimine (8), and finally, 1 is regenerated with the formation of L-threonine through a transaldimination reaction (see Figure 1(b)). In the side-reaction (6 → 1), 6 produces α-ketobutyrate via α-aminocrotonate, not the L-threonine. Kinetic analysis of the second-half reaction revealed that the phosphate ion produced in the first-half reaction promotes the main reaction in the second-half reactions.5 Therefore, ThrS represents a typical and straightforward example of product-assisted catalysis5,13, and what makes ThrS intriguing is that ThrS uses product-assisted catalysis as the basis of its reaction specificity. Then, it is necessary to elucidate the mechanism for the product-assisted catalysis by the phosphate ion. This elucidation is all the more important, since it will expand our knowledge on the function of this biologically important anion, the phosphate ion.

We have previously performed quantum mechanics/molecular mechanics (QM/MM) calculations to elucidate the catalytic reaction pathways for the product-assisted steps (6 → 7 → 8 → 1 rather than 6 → 1),14 and it was found that the phosphate ion stabilizes the transition state of the 6 → 7 reaction by forming a hydrogen bond with the hydroxyl group of the threonine moiety. However, such QM/MM calculations lack the sampling of conformational spaces for both the QM and MM regions. Indeed, in a plant ThrS, large conformational changes are observed on the active site upon the binding of its activator molecule.15 Furthermore, the plant TS exhibit disordered regions whose 3D structures are not determined due to their low electron density. According to the past theoretical reports,16,17 free energy contributions are known to be large for the QM–MM interactions in solvated systems. Therefore, in recent QM/MM free energy approaches, the major term can be evaluated by introducing an effective classical representation for the QM–MM



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interactions.16,17 Now, the molecular dynamics (MD) simulation is a practical approach to estimate the free energy contributions and to investigate the dynamic structures during the catalytic reactions.18 Consequently, a theoretical analysis based on MD will be favorable to deepen the understanding of the catalytic mechanism by taking into account the thermal fluctuations and structural changes. In the present study, MD simulations of ThrS were performed in order to elucidate the thermodynamically-stable active site structures in all the intermediate states in the main reaction of the second-half reaction (6, 7, 8). Formatted: Font color: Text 1

2. Computational details The crystal structure of Thermus thermophiles HB8 ThrS, complexed with an analog for PLP– α-aminocrotonate aldimine and a phosphate ion determined at a 2.1 Å resolution, was taken from the Protein Data Bank (PDB ID: 3AEX) and used as the starting structure.5 The AMBER force field (ff99SB-ILDN) and the general AMBER force field (gaff) were adapted for the protein and other molecules such as the substrate and ions, respectively. The Antechamber program of Amber12 was used to set up the non-standard molecules,19 and their atomic charges were transferred from the restrained electrostatic potential (RESP) charges calculated at the B3LYP/6-31G* theoretical level using the Gaussian 09 program package.20 After replacing an analog in the crystal structure to a proper substrate, the homodimer of ThrS was initially placed in a box filled with ~20,000 TIP3P water molecules (Figure S2 (A)). Six Na+ ions were added to neutralize the total system charge. The total number of atoms was ~70,000. All the MD calculations were run using the GROMACS 4.6 program.21 The electrostatic and Lennard-Jones (LJ) interactions were calculated using the particle-mesh Ewald (PME) with a short




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range cutoff of 10 Å and until a LJ cutoff of 9 Å is reached, respectively. Using the thermodynamic integration (TI) approach, the free energy differences depending on the phosphate and sulfate ions (∆G) were calculated using Eq. (1):

Δ = − =

= 〈

〉 = Δ

= 1 − +

: →

(1a)

(1b)

where the free energy difference ∆GAB was evaluated using the mixed potential V constructed by the linear product of two potentials, VA and VB. λ is the weight factor. For comparison to the experimental results, additional energy differences between the two intermediate states (∆∆G) were taken in the present study: ∆G = G(sulfate ion) – G(phosphate)

(2a)

∆∆G7−6 = ∆G7 – ∆G6

(2b)

where the index indicates the calculated state. For the evaluation of ∆∆G7−6 and ∆∆G8−7, six sets of ∆G calculations, three states (6, 7, 8) times 2 transitions (phosphate → sulfate and sulfate → phosphate), were performed. Thirteen λ values were used in this study λ = (0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 1). For each λ value, an energy minimization calculation with the steepest decent algorithm (hn = 0.01 nm, n = 50000 steps, ε = 100 kJ mol−1 nm−1) and 100 ps equilibrations using an NVT ensemble and NPT ensemble were performed. 3-ns NPT simulation was then performed and the trajectory was used for the free energy analysis. We calculated the round-trip pathways, λ = 0 → λ = 1 (phosphate → sulfate; up) and λ = 1 → λ = 0 (sulfate → phosphate; down). In order to accelerate the convergence of the free energy calculations, 15 different initial structures of ThrS (n = 15) were prepared from the MD trajectory and averaged for their TI results for the up and down pathways.22-24

Δ = ∑ Δ

: →

− Δ

: →

/2

(3)


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Therefore, the total simulation time length for the TI calculations was 3.51 µs (= 3 ns × 13 λ points × 15 repetitions × 2 pathway × 3 states). Computational details for the structural analysis are as follows. For the 6 states (3 states (6, 7 and 8) × 2 ions (phosphate or sulfate)), an energy minimization calculation with a steepest decent algorithm (hn = 0.01 nm, n = 10000 steps, ε = 1000 kJ mol−1 nm−1), a 50-ps equilibration using an NVT ensemble, and a 40-ps equilibration using an NPT ensemble were performed in order. 50-ns NPT simulations were then performed 10 times by changing the initial velocities, and their trajectories were used for the structural analysis. Therefore, for the structural analysis, the total MD time length was 3 µs (= 50 ns × 10 repetitions × 3 states × 2 ions). All the molecular structures depicted in this article were created using the visual molecular dynamics (VMD) program.25


3. Results and Discussion 3.1 Free energy differences between states and between divalent anions By using TI, the free energy differences between the phosphate and sulfate ions (∆G) were calculated for states 6, 7 and 8. In the TI methodology, thirteen λ points were selected to connect the different potentials of the phosphate ion (HPO42–) and sulfate ion (SO42–), and the free energy differences were evaluated by averaging their up (λ = 0 → λ = 1) and down (λ = 1 → λ = 0) pathways. All the calculated ∆G values are summarized in Table S1. By taking the additional difference of the ∆G values with respect to the states, the free energy double differences (∆∆G) were calculated to be ∆∆G7,6 = 1.09 kcal mol−1 and ∆∆G8,7 = −2.68 kcal mol−1. Based on the kinetic and spectroscopic analyses, the free energy profiles for the 6 →→ 1 steps were obtained.5 The free energy differences were ∆G7,6(P)exp. = 0.69 kcal mol−1 and ∆G8,7(P)exp. > 0.59 kcal mol−1 in the presence of phosphate ion, and ∆G7,6(S)exp. = 1.80 and ∆G8,7(S)exp. = 0.10 kcal



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mol−1 in the presence of sulfate ion. The P and S letters in parenthesis refer to the phosphate and sulfate ions, respectively. Therefore, the double free energy differences for the states and for the divalent anions are ∆∆G7,6exp. = 1.11 kcal mol−1, and ∆∆G8,7exp. < −0.49 kcal mol−1. The calculated ∆∆G values were in good agreement with the experimental results. Therefore, the present MD calculations are accurate enough to examine such small energy differences induced by the changes in the ions and states. The positive and negative ∆∆G values mean that the 6 → 7 transition is 1.1 kcal mol−1 more destabilized, while the 7 → 8 transition is more stabilized in the presence of the sulfate ion than in the presence of the phosphate ion. Although these energy changes are small, the energy differences reported for the transition states are more prominent compared to the energy differences between the intermediate states. That is, the activation energy of the 6 → 7 transition increased from ∆G7,6‡(P) exp. = G7,6‡(P) exp. – G6(P) exp. = 15.0 kcal mol−1 to ∆G7,6‡(S) exp. = 19.2 kcal mol−1, and those of the 7 → 8 transition decreased from ∆G8,7‡(P) exp. = 15.4 kcal mol−1 to a non-quantifiable value.5 Specifically, the differences for the activation barriers, ∆∆G7,6‡

exp.

= ∆G7,6‡(S)

exp.

–

∆G7,6‡(P) exp. = 4.2 kcal mol−1 and ∆∆G8,7‡ exp. < 0 kcal mol−1, are much increased compared to the differences in the intermediate states (∆∆G7,6exp. = 1.11 kcal mol−1, and ∆∆G8,7 exp. < −0.49 kcal mol−1). As there exist close energetic correlations between the intermediate and transition states, the detailed structural analyses for the intermediate states are performed in detail in the next sections. Compared to the transition states, for the intermediate states, it is much easier to calculate their structural properties based on theoretical approaches, such as using conventional classical MD methods. In the present study, MD methods were adapted to investigate the intermediate states, and their energetics and structural changes are precisely analyzed in order to elucidate the molecular mechanisms related to the reaction specificities.


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3.2. Conformational changes of PLP-substrate/product intermediate Based on the long time-scale MD simulations (total 3 µs), it was found that the side chain of the threonine moiety of PLP intermediate takes some specific conformations in states 7 and 8. By using two dihedral angles, φ = Angle[N‒Cα‒Cβ‒O] and ψ = Angle[Cα‒Cβ‒O‒H], the distributions of the PLP intermediate conformation were quantitatively classified using a clustering algorithm (FlexDice),26 and seven conformations (A–G) were identified (shown in Figures 2 and 3). Four conformations (A–D) were found in state 7, and three conformations (E–G) were found in state 8. The initial conformations in states 7 and 8 were conformations A and E, respectively. The probabilities for these conformations are summarized in Table 2. Their hydrogen bond types are (A) intramolecular hydrogen bond between the hydroxy and phosphate groups, (B) intramolecular hydrogen bond between the hydroxy and carboxy groups, (C, D) intermolecular hydrogen bond between the hydroxy group and the sulfate ion, (E) intramolecular hydrogen bond between the hydroxy and phosphate groups, (F) intramolecular hydrogen bond between the hydroxy and phosphate groups or intermolecular hydrogen bond between the hydroxy group and the sulfate ion or water molecule, and (G) intermolecular hydrogen bonds between the hydroxy group and the main chain O atom of Ala240 and between the hydroxy group of 8 and the hydroxy group of Thr85. The major conformations of state 7 with the phosphate ion (7P) are A and B, in which the phosphate ion always acts as a hydrogen bond donor and the hydroxyl group always acts as a hydrogen bond acceptor. In fact, the main conformation of 7P was A with the probability of 64.2%. The conformations of C and D are possible only for state 7 with the sulfate ion (7S), in which the hydroxy group becomes the hydrogen bond donor to the carboxy group. Nevertheless, for 7S, conformations C and D are minor due to their steric hindrances. As a result, the main conformation of 7S is B (46.5%). In state 8, due to the protonation of Cα, this C atom becomes sp3-hybridized, causing the




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conformational change in the side chain of the threonine moiety of the intermediate to allow the dihedral angles of φ = 60° and ‒60°. The major conformations for state 8 with the phosphate ion (8P) and state 8 with the sulfate ion (8S) are G and F, respectively. As the main conformation of 7P was conformation A, it was considered that the initial conformation E, which is the direct descendant of conformation A, was changed to conformations F and G. We found that conformation F favors the formation of the characteristic three hydrogen bonds connecting a water molecule to the phosphate group, the hydroxyl group, and phosphate ion in a Y-shaped manner (Figure 4). These Y-shaped hydrogen bonds are formed in 64.5% of conformation F (39.2%) in 8P, and 63.6% of F (82.9%) in 8S. This suggests that the Y-shaped hydrogen bonds are the major form in conformation F. As the main conformation of 8S is F, the sulfate ion appears to prevent to taking conformation G, which is the main conformation in 8P. For the stabilization of conformation F in 8S, the ∆∆G8,7 value will become negatively high.

3.3 Role of the divalent anion for the PLP–substrate/product intermediates In order to analyze the interactions between the divalent anion (phosphate/sulfate ion) and the PLP–substrate/product intermediates, the radial distribution between the oxygen atom of the hydroxy group of 7 or 8 and the closest oxygen atom of the divalent anion were plotted in Figure 5. In 7P, there is a single peak with a maximum at 2.9 Å and almost all the distributions are within the hydrogen bond distance of 3.2 Å. This indicates the formation of a rigid hydrogen bond between the phosphate ion and the hydroxy group in conformation A or B, where there exist additional hydrogen bonds between the hydroxy group and phosphate group or carboxy group. In 7S, two peaks are observed at 2.6 Å and 3.2 Å. The closer peak comes from conformations C and D, in which the



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sulfate ion forms a hydrogen bond with the hydroxy group. On the other hand, the farther peak comes from conformations A and B, in which the hydrogen bonds are not formed because the sulfate ion only acts as a hydrogen bond acceptor. Therefore, these results clearly suggest that the


role of the phosphate ion is the formation of the hydrogen bond with the hydroxy group of 7. In 8P, two peaks are observed. The first peak exists at around 2.8 Å and the second peak exists at around 5.3 Å. The former peak comes from two conformations, E and F. For this peak, the phosphate ion and the hydroxy group in conformation E act as the donor and acceptor, respectively, and they become the acceptor and donor, respectively, in conformation F. The latter peak is attributed to conformation G and a hydrogen bond is almost negligible for this long distance. In 8S, two peaks are observed at around 2.6 Å and at around 5.3 Å. Both peaks come from conformation F. For the peak at around 2.6 Å, the sulfate ion and the hydroxy groups are a hydrogen bond acceptor and donor, respectively, and for the peak at 5.3 Å, one water molecule is involved between the sulfate ion and PLP-L-threonine aldimine in the Y-shaped form (Figure 4). Therefore, in the 8S state, both peaks indicate strong hydrogen bonds between the sulfate ion and the hydroxy group of 8.

3.4 Interactions between the divalent anion and water molecules During the MD simulations, water molecules are inserted into the active site and are close to the divalent anion (phosphate/sulfate ion). These water molecules will be related to the structural stability and catalytic reactions. Therefore, the radial distributions between the central atom of the divalent anion (P atom of the phosphate ion or S atom of the sulfate ion) and oxygen atom of the water molecules are plotted in Figure 6. In state 7, no significant differences are observed between 7P and 7S regarding the water distributions. In states 6 and 8 with the sulfate ion, however, more




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water molecules are inserted into the active site compared to the states with the phosphate ion. Therefore, the phosphate ion has a possible role to regulate the behavior of the water molecules, depending on the active site state. As the phosphate ion exists at the entrance position, the ion acts like a gatekeeper for water molecules around the active site of ThrS. The present water distribution changes in relation to the structural changes in the amino acids near the active site and the protein are discussed in the next two sections.

3.5 Conformational changes of the Lys61 side chain In our previous QM/MM study, Lys61 was found to be the catalytic base and acid for the 6 → 7 and 7 → 8 reactions, respectively.14 In the 8 → 1 reaction, the Lys61 side chain reacts with the PLP–L-threonine aldimine, undergoing a transaldimination reaction. In spite of these crucial roles of Lys61 in ThrS, the most stable conformation of the Lys61 side chain has not been clearly revealed by the QM/MM calculations, because large conformational freedoms are expected for its long side chain, and the feasible positions of the Lys61 amine during the catalytic steps may be different from step to step. Therefore, the stable conformations of the Lys61 were analyzed based on the present MD trajectories. Contrary to our expectation, only two conformations were found for the Lys61 side chain (Figure 7). One is the conformation with the amino group of Lys61 hydrogen bonded to the phosphate/sulfate ion (Figure 7a), and the other with the amino group of Lys61 hydrogen bonded to the PLP phenolate oxygen (O3′; Figure 7b). The former conformation is dominant, and the latter is minor and only observed for 6S, 8P and 8S. In 6P, 7P and 7S, the latter conformation is not observed. In the present study, we call the latter conformation the “slide conformation”. The


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probability of the Lys61 slide conformation for each state is summarized in Table 3. In state 7, the protonated form of the amino group of Lys61 forms a rigid salt bridge with the phosphate/sulfate ion or the PLP phosphate group. As the phosphate/sulfate ion in state 7 is stabilized in the active site with the hydrogen bond by the hydroxyl group of PLP-L-threonine, the major conformation of the Lys61 side chain is more favorable compared to the slide one. The slide conformation of Lys61 was observed at the probability of 6.0% and 3.8% in states 8P and 8S, respectively. In the slide conformation, the PLP–substrate/product complex is almost in conformation G (80% is in conformation G and 20% is in conformation F). In 8S, the slide conformation is observed in conformation G. Therefore, the slide conformation of Lys61 is favorable in conformation G, which is specific to 8. In 6S, the slide conformation of Lys61 can be observed with a very minor probability (0.3%). The slide conformation of Lys61 is suitable for the transaldimination reaction (8 → 1 and 6 → 1) which involves the direct nucleophilic attack of the amino group of Lys61 on the C4′ atom of PLP. This 8P has a significant probability of conformation G (6.0%) which is consistent with the fact that ThrS catalyzes the formation of L-threonine

in the presence of phosphate ion.

An experimental kinetic analysis showed that the sulfate ion in place of the phosphate ion drives 6 to the side reaction rather than the main reaction.5 The side reaction from state 6 is a transaldimination reaction to form α-aminocrotonate, which is non-enzymatically hydrolyzed to α-ketobutyrate. The presence of conformation G in 6S and the absence of it in 6P explain the fact that ThrS preferentially catalyzes the side reaction in the presence of the sulfate ion. Thus, the populations of the slide form of Lys61 in states 6S, 8P and 8S, obtained in the present MD results, reasonably explain the differential behavior of the phosphate and sulfate ions in controlling the reaction specificity of ThrS.



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3.6 Flipped structure of Phe134 As discussed in sections 3.2 and 3.4, the water molecules in the catalytic site play critical roles in the stabilization of the states. In 8S, one water molecule comes into the catalytic site and forms the Y-shaped hydrogen bonding network, thereby stabilizing the complex structure. In the calculated six states (6P, 6S, 7P, 7S, 8P, and 8S), bulk solvent water molecules are hydrogen bonded to the divalent anions. By observing the trajectories of these water molecules, we found that these water molecules come through the cavity that is covered by an α-helix (a6, Asn133–Phe148), which is responsible for the open-closed conformational change in ThrS. It should be noted that both the open- and closed-structures are solved in ThrS (PDB IDs are 1UIM and 3AEX for open and closed structures, respectively).5,27 The current MD simulations are not long enough to cause a transition to the open structure involving the movement of the α-helix a6. However, a flipped structure of Phe134 could be observed in the present study. As Phe134 locates at the N-terminal position of the α-helix, this structure is expected to be a prerequisite for the ejection of the product and inclusion of bulk water molecules. Phe134 is a highly conserved residue in the ThrS proteins from various sources. In the closed-form of the X-ray structure, the side chain of Phe134 is located in a hydrophobic region surrounded by the Ala83, Leu138, Ile243 and methyl group (Cγ) of the substrate/product moiety of the intermediates. In the flipped structure obtained in this MD study, the side chain of Phe134 locates in another hydrophobic region surrounded by the side chains of Ala83, Val106, Pro108, Val130 and Ala137. The distributions of Phe134 are analyzed by taking the probabilities of the distance between the Cα atom of the intermediate and the Cζ atom of Phe134 (Figure 8). In the closed structure, the distance is about 6 Å, and in the shifted state, the distance increased to about 8 Å. Figure 8 shows


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that the Phe134 flip is obviously observed in states 7P and 7S. The flips are significantly suppressed in states 6P and 6S, and are suppressed to about half in states 8P and 8S, compared to the states 7P and 7S. Therefore, depending on the state of the active site, the mobility of the Phe134 is different. It is interesting to note that the present MD simulation suggests that water molecule(s) come into the active site not at the intermediate 6, but after intermediate 7. That is, it is considered that a water molecule present at the active site is used for the nucleophilic attack on 6 to form 7, and then a water molecule is replenished from the solvent.


4. Conclusions In the present study, MD-based theoretical analyses were performed in order to elucidate the molecular mechanism on the product-assisted catalysis of ThrS; phosphate ion drives the main reaction to produce L-threonine (6 → 7 → 8 → 1), while the sulfate ion, a divalent anion analogous to phosphate ion, promotes the side reaction (6 → 1) leading to the formation of α-ketobutyrate. We performed two sets of MD simulations. Free energy evaluations using the thermodynamic integration approach (simulation time length to be 3.5 µs) gave ∆∆G values (change in the relative energy levels of the intermediates 6, 7, and 8 caused by the substituting sulfate for phosphate ion) that are in good agreement with the experimental results. A 3-µs MD calculation of the intermediate states 6, 7, and 8, in the presence of phosphate ion or sulfate ion, was then performed in order to calculate the populations of the conformation of the intermediates, including the positions of the water molecules, formations of hydrogen bonds, and conformations and positions of the important amino acids (Lys61, Phe134). An interesting finding was that in 8 with the sulfate ion (8S), one water molecule comes from the bulk water and forms a



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Page 16 of 35

Y-shaped hydrogen bond network with the hydroxy group and the phosphate group of PLP and the sulfate ion. This structure is formed in conformation F of the intermediate, which is the preferable conformation in 8S compared to 8P, accounting for the relative stabilization of 8S (∆∆G 8,7 < 0). From the radial distribution of water molecules around the divalent anion, more water molecules come into the active site in 6 and 8 with the sulfate ion. No differences in the water distribution with respect to the divalent ions were observed in 7. Based on the present MD results, the roles of the phosphate ion for the product-assisted catalysis of ThrS are summarized as follows. The phosphate ion suppresses the incoming of water molecules especially in 6 and 8 in order to maintain the rigid packing of the intermediate in the active site. In 7, the major conformations were A and B, in which the phosphate ion and hydroxy group of the intermediates are strongly hydrogen bonded. In 8, conformation G is more populated compared to conformation F. Conformation G is closely related to the slide conformation, in which the amino group of Lys61 hydrogen-bonds to the phenolic O of PLP and is at the favorable position for the nucleophilic attack on C4′ of PLP. Both the relatively elevated free energy level and the more populated conformation G of 8P, compared to 8S, are considered to contribute to the efficient


release of the product via the transaldimination reaction. On the other hand, in 6, the slide conformation was completely absent in the presence of phosphate ion, although it was observed in the presence of sulfate ion, accounting for the finding that phosphate ion suppresses the side reaction starting from 6 via transaldimination observed in the presence of sulfate ion. Thus, the phosphate ion in the active site of ThrS properly controls the conformation of the intermediates suitable for the progress of the main reaction. It is noteworthy that the conformational controls are performed by changing the hydrogen bond interactions around the intermediates. It was also clearly shown that the phosphate ion plays a key role in the distributions of water molecules as well as for the hydrogen bond formation involving them. The reaction control by the phosphate ion



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is not energetically strong (~ 5 kcal mol−1), but the fine tuning of the substrate conformations and hydrogen bonds in the active site will be practically important as the critical molecular mechanism revealed in the present study. We would like to emphasize again that, in addition to the formation of the hydrogen bond between the

phosphate

ion

and

the

transition

states,

controlling

the

conformation

of

the

PLP-substrate/product intermediates and amino acid residues in the active site is one of the important strategies for the phosphate ion to promote the main reaction in ThrS. Thus, the phosphate ion has multiple important functional roles in the product-assisted catalysis of ThrS. Discrimination between the phosphate ion and the sulfate ion is a fundamental problem in biophysical chemistry, and the molecular mechanism elucidated here would have significance in studying this problem. Finally, it is important to point out that the TI methodology can well reproduce the very small free energy differences (~1 kcal mol-1) between the phosphate and sulfate ions. This success was made possible by the long time-scale MD and the relatively limited structural changes for a sequence of the intermediate states and divalent anions. As the present theoretical approach was shown to be very practical and invaluable for elucidating the characteristic structural changes in a multistep reaction, further applications to other multistep reactions are expected.




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Associated Content Supporting Information

Notes The authors declare no competing financial interests. Formatted: Font color: Text 1

Acknowledgements This research was supported by the Grant-in-Aid for Scientific Research (C; No. 26410002) from the Japan Society for the Promotion of Science. Numerical calculations have been carried out under the supports of (1) HPCI system research project (Project ID: hp160169) using the computational resource of the center for computational sciences (CCS), University of Tsukuba, (2) “Interdisciplinary Computational Science Program” at CCS. Formatted: Font color: Text 1


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References 1 Laber, B.; Gerbling, K.-P.; Harde, C.; Neff, K.-H.; Nordhoff, E.; Pohlenz, H.-D. Mechanisms of interaction of Escherichia coli threonine synthase with substrates and inhibitors. Biochemistry 1994, 33, 3413-3423. 2 Hayashi, H. Pyridoxal enzymes: mechanistic diversity and uniformity. J. Biochem. 1995, 118, 3, 463-473. 3 Garrio-Franco, M.; Ehlert, S.; Messerschmidt, A.; Marinkovic, S.; Huber, R.; Laber, B.; Bourenkov, G. P.; Clausen, T. Structure and Function of Threonine Synthase from Yeast. J. Biol. Chem. 2002, 277, 14, 12396-12405. 4 Covarrubias, A. S.; Högbom, M.; Bergfors, T.; Carroll, P.; Mannerstedt, K.; Oscarson, S.; Parish, T.; Jones, T. A.; Mowbray, S. L. Structural, Biochemical, and In Vivo Investigations of the Threonine Synthase from Mycobacterium tuberculosis. J. Mol. Biol. 2008, 381, 622-633. 5 Murakawa, T.; Machida, Y.; Hayashi, H. Product-assisted Catalysis as the Basis of the Reaction Specificity of Threonine Synthase. J. Bio. Chem. 2011, 286, 4, 2774-2784. 6 John, R. A. Pyridoxal phosphate-dependent enzymes. Biochimica et Biophysica Acta 1995, 1248, 81-96. 7 Percudani, R.; Reracchi, A. A genomic overview of pyridoxal-phosphate-dependent enzymes. EMBO reports 2003, 4, 850-854. 8 El-Sayed, A. S.; Shindia, A. A. PLP-dependent enzymes: a potent therapeutic approach for cancer and cardiovascular diseases. Targets in Gene Therapy, Prof. Yongping You (Ed.), InTech, 2011. Chapter 7. 40. DOI: 10.5772/17449. 9 Toney, M. D. Controlling reaction specificity in pyridoxal phosphate enzymes. Biochimica et Biophysica Acta 2011, 1814, 1407-1418. 10 Salvo, M. L. DI; Budisa, N.; Contestabile, R. PLP-dependent enzymes: a powerful tool for



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metabolic synthesis of non-canonical amino acids. Proceedings of the Beilstein Bozen Symposium on Molecular Engineering and Control. 2012, 27-67. 11 Morneau, D. J. K.; Abouassaf, E.; Skanes, J. E.; Aitken, S. M. Development of a continuous assay and steady-state characterization of Escherichia coli threonine synthase. Anal. Biochem. 2012, 423, 78-85. 12 Gahungu, M.; Arguelles-Arias, A.; Fickers, P.; Zervosen, A.; Joris, B.; Damblon, C.; Luxen, A. Synthesis and biological evaluation of potential threonine synthase inhibitors: Rhizocticin A and Plumbemycin A. Bioorg. Med. Chem. 2013, 21, 4958-4967. 13 Fromme, J. C.; Bruner, S. D.; Yang, W.; Karplus, M.; Verdine, G. L. Product-assisted catalysis in base-excision DNA repair. Nat. Struct. Biol. 2003, 10, 204-211. 14 Shoji, M.; Hanaoka, K.; Ujiie, Y.; Tanaka, W.; Kondo, D.; Umeda, H.; Kamoshida, Y.; Kayanuma, M.; Kamiya, K.; Shiraishi, K.; Machida, Y.; Murakawa, T.; Hayashi, H. A QM/MM Study of the L-Threonine Formation Reaction of Threonine Synthase: Implications into the Mechanism of the Reaction Specificity. J. Am. Chem. Soc. 2014, 136, 12, 4525-4533. 15 Mas-Droux, C.; Biou, V.; Dumas, R. Allosteric threonine synthase reorganization of the pyridoxal phosphate site upon asymmetric activation through s-adenosylmethionine binding to a novel site. J. Biol. Chem. 2006, 281, 8, 5188-5196. 16 Valiev, M.; Garrett, B. C.; Tsai, M.-K.; Kowalski, K.; Kathmann, S. M.; Schenter, G. K.; Dupuis, M. Hybrid approach for free energy calculations with high-level methods: Application to the SN2 reaction of CHCl3 and OH− in water. J. Chem. Phys. 2007, 127(5), 051102-4. 17 Duarte, F.; Amrein, B. A.; B-Nelson, D.; Kamerlin, S. C.L. Recent advances in QM/MM free energy calculations using reference potentials. Biochim. Biophys. Acta 2015, 1850, 954-965. 18 Tanaka, W.; Shoji, M.; Tomoike, F.; Ujiie, Y.; Hanaoka, K.; Harada, R.; Kayanuma, M.; Kamiya, K.; Ishida, T.; Masui, R.; Kuramitsu, S.; Shigeta, Y. Molecular mechanisms of substrate


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specificities of uridine-ctydine kinase. biophysics and physicobiology 2016, 13, 77-84. 19 Case D. A. et al., AMBER 12, University of California, San Francisco, 2012. 20 Frisch M. J. et al., Gaussian 09, Revision D.01, Gaussian, Inc., Wallingford CT, 2013. 21 Spoel, D. van der; Lindahl, E.; Hess, B. and the GROMACS development team, GROMACS User Manual version 4.6.5, www.gromacs.org 2013. 22 Hummer, G. Fast-growth thermodynamic integration: Error and efficiency analysis. J. Chem. Phys. 2001, 114, 17, 7330-7337. 23 Suenaga, A.; Umezu, O.; Ando, T.; Yamato, I.; Murata, T.; Taiji, M. Estimation of Ligand Binding Free Energies of F-ATPase by Using Molecular Dynamics/Free Energy Calculation. J. Comput. Chem. Jpn. 2008, 7, 3, 103-116. 24

Lawrenz,

M.;

Baron,

R.;

McCammon,

J.

A.

Independent-Trajectories

Thermodynamic-Integration Free-Energy Changes for Biomolecular Systems: Determinants of H5N1 Avian Influenza Virus Neuraminidase Inhibition by Peramivir. J. Chem. Theory Comput. 2009, 5, 4, 1106-1116. 25 Humphrey, W.; Dalke, A.; Schulten, K. J. VMD: Visual molecular dynamics. Molec. Graphics 1996, 14, 33-38 . 26 Nakamura, T.; Kamidoi, Y.; Wakabayashi, S.; Yoshida, N. Information Proceeding Society of Japan, Journal Database 2005, 46, 40-49. 27 Omi R.; Goto, M.; Miyahara, I.; Mizuguchi, H.; Hayashi, H.; Kagamiyama, H.; Hirotsu. H. Crystal structures of threonine synthase from Thermus thermophilus HB8: conformational change, substrate recognition, and mechanism. J. Biol. Chem. 2003, 14, 278(46), 46035-46045.



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Figure Captions

Figure 1. (a) Structure of threonine synthase (ThrS) (PDB ID:3AEX). (b) Schematic representation of the active site in the second-half reaction (6 → 7 → 8). The phosphate ion near the PLP-substrate/product intermediate is produced in the first-half reaction (4 → 5), and the

Formatted: Font color: Text 1 Formatted: Font color: Text 1 Formatted: Font color: Text 1

phosphate ion aides in the latter main catalytic reactions. Enlarged views for the active site of ThrS are shown in Figure S2 (B-D).

Figure 2. Probability distributions of the conformations of the PLP intermediates. φ and ψ are the dihedral angles defined for N–Cα–Cβ–O and Cα–Cβ–O–H, respectively. The net probability for each conformation is summarized in Table 2. States 7P and 7S and 8P and 8S indicate states 7 and 8 with phosphate and sulfate ions, respectively.

Figure 3. Representative structures of the conformations of the PLP intermediates (A – G). Conformations A – D appear in state 7, and conformations E – G appear in state 8. Representative structures in these conformations are provided in the PDB file format via the ACS website (see Table S3, supporting information).

Figure 4. The hydrogen bonds between PLP intermediate and water molecule observed in state 8 with sulfate ion (8S).

Figure 5. Radial distribution functions between the oxygen (O3’) atom of the PLP pyridoxal ring Formatted: Font color: Text 1

and closest oxygen atom of the divalent anion.


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Figure 6. Radial distribution functions (RDF) between the oxygen atom of the water molecules and the central atom of the divalent anion (P and S atoms for phosphate and sulfate ions, respectively).


Figure 7. (a) X-ray structure of the active site and (b) flipped structure of Lys61 observed in state 8P. (a) shows the major conformation of the Lys61 side chain which forms hydrogen bonds with the PLP phosphate group and divalent ion. (b) shows the minor conformation of the Lys61 side chain which forms a hydrogen bond with the oxygen atom (O3’) of the PLP pyridine ring.

Figure 8. Distribution function of the distance between Cα atom of PLP intermediate and Cζ atom of Phe134. Peaks around 8 Å correspond to the Phe134-shifted structures.




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Page 24 of 35



Fig. 1. Y. Ujiie et al.


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E

A P

φ

ψ B

P

B

ψ

F G

ψ φ

φ

State 7P

State 8P

A

E

D C

ψ

ψ B

B

F G

D

φ

φ

State 7S

State 8S




1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



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P

S




1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Lys61

Lys61

P

C4’

O3’

P

P

P

P

(a)

(b)



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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table 1. Calculated double differences of the free energy (∆∆G/ kcal mol−1) between states and the ions of ThrS.a ∆∆G7–6

∆∆G8–7

Calc.

1.09

–2.68

5

1.11

< -0.49

Exp. a

∆∆G7–6 = ∆G(state 7) – ∆G(state 6), ∆G(state 6) = G(state 6, SO42–) – G(state 6, HPO42–)


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Table 2. Probability distributions (%) of conformations Ion

State 7

State 8

A

B

C

D

E

F

G

HPO42−

64.2

33.8

–

–

10.5

39.2

46.9

SO42−

25.1

46.5

3.8

20.0

5.7

82.9

8.1



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Table 3. Probability (%) of the slide conformation of the side chain of Lys61 State 6

State 7

State 8

HPO42−

0

0

6.0

SO42−

0.3

0

3.8


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most stable conformations in intermediate states 89x66mm (300 x 300 DPI)


Molecular Mechanism of the Reaction Specificity in Threonine Synthase

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