Insights into the Thiamine Diphosphate Enzyme ... - ACS Publications

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Insights into the Thiamine Diphosphate Enzyme Activation Mechanism: Computational Model for Transketolase Using a Quantum Mechanical/Molecular Mechanical Method Lionel Nauton,†,‡ Virgil Hélaine,†,‡ Vincent Théry,*,†,‡ and Laurence Hecquet*,†,‡ †

Université Clermont Auvergne, Université Blaise-Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448, F-63000 Clermont-Ferrand, France ‡ CNRS, UMR 6296, ICCF, F-63178 Aubiere, France S Supporting Information *

ABSTRACT: We propose the first computational model for transketolase (TK), a thiamine diphosphate (ThDP)-dependent enzyme, using a quantum mechanical/molecular mechanical method on the basis of crystallographic TK structures from yeast and Escherichia coli, together with experimental kinetic data reported in the literature with wild-type and mutant TK. This model allowed us to define a new route for ThDP activation in the enzyme environment. We evidenced a strong interaction between ThDP and Glu418B of the TK active site, itself stabilized by Glu162A. The crucial point highlighted here is that deprotonation of ThDP C2 is not performed by ThDP N4′ as reported in the literature, but by His481B, involving a HOH688A molecule bridge. Thus, ThDP N4′ is converted from an amino form to an iminium form, ensuring the stabilization of the C2 carbanion or carbene. Finally, ThDP activation proceeds via an intermolecular process and not by an intramolecular one as reported in the literature. More generally, this proposed ThDP activation mechanism can be applied to some other ThDP-dependent enzymes and used to define the entire TK mechanism with donor and acceptor substrates more accurately.

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or Campylobacter jejuni and from humans has been determined, revealing high degrees of homology with the active site of yeast TK.16−20 In addition, TK is an enzyme of great interest in various domains such as biocatalysis for asymmetric synthesis of enantiopure ketoses by carboligation,21−29 and a target for the prevention and treatment of various cancers, neurodegenerative diseases, and diabetes, offering new prospects in structure-based drug design.30−32 In the ThDP-dependent enzyme mechanism described, the reactive C2 atom of the ThDP thiazolium ring attacks the carbonyl of the donor substrate to form a ThDP adduct. Prior to this reaction, ThDP has to be activated. The mechanism for ThDP activation in yeast TK has been investigated using crystallographic, near-UV circular dichroism (CD) and nuclear magnetic resonance (NMR) data, and in kinetic studies with wild-type TK and single-point mutants in the presence of natural substrates and analogues. In the yeast TK active site (Scheme 1),33−36 ThDP activation starts with a proton transfer (step a) from Glu418B to N1′ of the aminopyrimidine ring, giving intermediate 2. The N4′ amino group is converted into a N4′ iminium group (intermediate 3) by a subsequent

hiamine diphosphate (ThDP)-dependent enzymes form a vast, diverse class of proteins, catalyzing a broad variety of enzymatic reactions, including the formation or cleavage of carbon−sulfur, carbon−oxygen, carbon−nitrogen, and especially carbon−carbon bonds.1−4 These properties are of great interest for the chemoenzymatic synthesis of various compounds.5−8 For novel therapeutic approaches, ThDP-dependent enzymes of human origin have been identified as being involved in a variety of diseases.9 Although very diverse in sequence and domain organization, these enzymes share two common protein domains, the pyrophosphate and the pyrimidine domains, which have similar structures and are essential for binding and activating ThDP. The mechanism of ThDP-dependent enzymes has been extensively studied using different spectroscopic techniques on various enzymes.10−14 ThDP activation is the initial and crucial reaction common to all ThDP-dependent enzymes and has been the subject of controversy over the past 30 years. Of the ThDP-dependent enzymes, transketolase (TK, EC 2.2.1.1) is one of the most extensively studied, judging by the abundant data available in the literature. TK is therefore a highly useful model for investigating the ThDP activation process. The three-dimensional structure of TK from yeast in the native form and cocrystallized with donor or acceptor substrates was first studied.15 Recently, the three-dimensional TK structure from other microorganisms such as Escherichia coli © XXXX American Chemical Society

Received: July 15, 2015 Revised: March 7, 2016

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DOI: 10.1021/acs.biochem.5b00787 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry Scheme 1. ThDP Activation Mechanism for Yeast TK Described in the Literature

mesomeric effect (step b). Deprotonation of this iminium group by His481B then leads to the corresponding N4′ imino group in intermediate 4 (step c). This imino group can later act as an intramolecular base for C2 proton abstraction to give the ylide-ThDP 5 (step d), giving back the N4′ iminium moiety. In the final step (e), Glu418B abstracts the N1′ proton, leading, after a mesomeric effect, to the active form of ThDP 6, which can react with the TK donor substrate. However, as mentioned above, this proposed mechanism has some failings. First, the study of TK crystal structures available in the Protein Data Bank (PDB) reveals that the only close basic residue able to abstract the N4′ proton from the iminium group in intermediate 3 (step c, Scheme 1) is His481B, as stated by Schneider et al.37 Although the distance between His481B Nε and N4′ (3.05 Å) would allow such proton abstraction, a hydrogen bond between His481B Nδ and the oxygen (Og1) atom of Thr480B holds His481B in an unfavorable orientation. In addition, proton exchange NMR experiments conducted with the His481Ala mutant show that C2 deprotonation still occurs,33 albeit with a dramatic drop in TK activity. These findings indicate that His481B may be a crucial residue for TK catalytic activity, but not for ThDP activation. This is illustrated in other TK sources such as human TK19 and in other ThDPdependent enzymes such as benzaldehyde lyase38 or glyoxylate carboligase,39 where ThDP activation was efficient although His481B is missing, with no other His near ThDP or too far from N4′. In all cases, the question of how the C2 proton is abstracted from N4′ iminium arises. The nature of the base must be determined, because this is essential for the tautomerism between states 1 and 4 (Scheme 1). Second, during step d (Scheme 1), as seen above, ThDP is activated by an intramolecular proton transfer from the C2 thiazolium ring to the N4′ imino group, giving intermediate 5. In this hypothesis, the σ* orbital of the C2−H bond should be aligned with the lone pairs of the N4′ imino group. However, because of the well-known V-shape of ThDP in TK (Figure 1), the angle between the plane defined by S1, C2, and N3 and the

Figure 1. Thiamine diphosphate in a V-shape from the structure of Protein Data Bank entry 1TRK.

plane defined by N3′, C4′, and N4′ is 74.7°, far from the theoretical value of 180° required for an energetically feasible transfer. Also, in density functional theory (DFT) studies40−42 conducted on ThDP ylide formation, Lie and Schiott40 studied ThDP ylide formation in pyruvate decarboxylase (PDC) with implicit, explicit, or mixed solvation models and showed that the iminium form is not stable in a V-shape (Φp = −90.0°; Φt = 90°) but rather in an F-shape (Φp = −76.9°; Φt = 1.8°).40 Because of the ThDP V-shape mentioned above, these data led the authors to consider that ThDP activation might be achieved via an imino group, in the presence of the enzyme. However, this study was conducted with no enzyme, and the authors gave no information about the deprotonation route of the iminium function in this putative imino moiety. We note that the authors40 stated, in 2008, that the direct or water-mediated proton transfer from C2 to N4′ is energetically favorable. This hypothesis was also suggested in 2013 by Tittmann and colleagues46 for pyruvate oxidase. B


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Figure 2. Overview of the two ONIOM layers (green sticks, HIGH layer; red sticks, LOW layer with free atoms; wire, LOW layer with frozen atoms).

Third, the mechanism of ThDP deprotonation could also be explained by taking into account pKa values of the active centers (C2, N4′, and N1′) of ThDP that were considered. However, local pKa values within the active site are difficult to assess precisely as shown by the range of pKa values found (e.g., for C2, from 6 to 17−19).43−46 Fourth, this known mechanism is still today considered as universal regardless of what enzymatic reactions or amino acids are involved, except for Glu418B, which is highly conserved. Thus, most published experimental results have been interpreted according to this mechanism. Other elements involved in the mechanism have been mentioned, such as the Lewis acid role of the N4′ iminium group, by Sable and Meschke46 but not further considered. On the basis of crystallographic TK structures from yeast and E. coli available in the PDB (1TRK, 1GPU, 1AY0, 1QGD, 2R5N, 2R8O, and 2R8P)28,30,48,49 and experimental data obtained by kinetic studies with wild-type and mutant TK, we propose the first computational model for yeast TK using a quantum mechanical/molecular mechanical (QM/MM) method. With this model, we investigated a new activation pathway for ThDP in the enzyme environment.

The results of the complete ThDP activation reaction path with the transition state (TS) were validated by the intrinsic reaction coordinate (IRC). The initial and final complexes, named IRC reactant and IRC product, respectively, were obtained by minimizing the forward and reverse IRC points. A frequency calculation was conducted to check the minima and the transition state. The first set of QEQ charges was defined for the initial model (Figure 2) and then fixed for all the calculations with the 6-31G basis set to avoid energy differences between each state, preventing their comparison. Every cut between the HIGH layer and the LOW layer was defined on a σ bond. Design of the Model Using a Two-Layer ONIOM Method. The model was designed from the 1TRK structure, the native form of yeast TK, and was focused on the active site identified by ThDP from the A chain. The hydrogens were automatically added. Thus, all residues were protonated automatically except for His residues, which were manually protonated depending on their abilities to form an H-bond with their immediate surroundings. His30A, His69A, His103A, and His481B of the active site were thus protonated on Nδ and on Nδ,ε for His263A. Glu418B and Glu162A were also manually protonated. Our model was built by including every amino acid involved in ThDP activation or stabilization. The following amino acids (A chain) were included: Gly29A−Met37GlyA (for the sake of calculation convenience, some amino acids have been replaced), Leu65A−Tyr75A, Phe93A−Glu105A, Thr115A−Gly121A, Leu155A−Ile164A, Asp185A−Ile191A, Thr248A−Gly251A, and Ala257A−Ala265A. For the B chain, the following amino acids were included: Gly163B−Leu171B, Gly379B−Ser386B, Tyr414B−Ile424GlyB, Gly439B−Ala449B, and Trp465B−Pro483B. All N-terminal and C-terminal positions were set in their neutral form. Finally, the model

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MATERIALS AND METHODS Computational Strategy. All computations were performed with GAUSSIAN 09 revision D0350 and the two-layer ONIOM51,52 method with mechanical and electronic embedding. For the “LOW” layer, a classical mechanics force field level UFF53 was applied with QEQ charges.54 For the “HIGH” layer, the density functional theory55,56 (DFT) level with B3LYP57−60 hybrid functional and 6-31G basis sets were applied. Energy minimizations were performed with the Quadmac option.61 C


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addition, H2O688A was also involved in an H-bond with the ThDP C2 proton (Table 1 entry 4a). These results indicate a putative role of this water molecule in ThDP deprotonation as a bridge between C2 and Nδ of His481B. We also observed a short H-bond showing a strong interaction between ThDP N1′ and Glu418B H-O (Table 1, entry 11a), but with no hydrogen transfer. However, this strong interaction is sufficient to induce a mesomeric effect in the pyrimidine cycle and not a tautomeric one. The short C4′−N4′ distance (Table 1, entry 9a) clearly indicates a π delocalization toward an N4′ state close to sp2 hybridization (Table 2 entry 4a) corresponding to an iminium group. Interestingly the HN4′ proton interacts via an H-bond with H2O688A (Table 1, entry 6a), but not with His481B, as reported in the literature. New Reaction Pathway for ThDP Activation. Structural Aspect. From this minimized structural model, we investigated the reaction pathway by stretching the C2−H bond. This scan gave a putative transition state corresponding to a 1.33 Å C2− H bond distance (corresponding to the maximal energy of the scan). We performed TS optimization (Figure 4) followed by IRC (Figure S1). Both structures obtained after 20 points in forward and reverse directions were minimized at the B3LYP/ 6-31G level, taking into account thermochemistry corrections at 298.15 K and 1.0 atm. IRC reactant [inactivated ThDP (Figure 5A)] and IRC product [activated ThDP (Figure 5B)], respectively, were obtained. A comparison of both bond lengths and angle values obtained in the model (Tables 1 and 2, entry a) and in the IRC reactant (Tables 1 and 2, entry b) revealed no significant differences, validating our model. In the literature (Scheme 1), ThDP activation starts with a proton transfer from Glu418B to ThDP N1′ (step a). In our proposed mechanism, the proton originally located on Glu418B in IRC reactant (Table 1, entries 10b and 11b) is found almost equidistant from N1′ and O Glu418B (Table 1, entries 10d and 11d) in the IRC product. Hence, although the interaction between N1′ and HO Glu418B is very strong [1.45 Å (Table 1, entry 11b)], the proton is not transferred from Glu418B to ThDP N1′, whereas (Table 1, entries 11c and 11d), the distance between N1′ and O Glu418B remained unchanged (Table 1, entries 12b and 12d). Nevertheless, our mechanism is consistent with the mesomeric effect reported in the literature (Scheme 1, step b) because the C4′−N4′ bond length (Table 1, entry 9) is on the order of magnitude of that of a double bond and so concordant with an iminium moiety. We note that both C4′−N4′ bond length and N4′ atomic charge remained unchanged upon comparison of IRC product and reactant. These data underline the strong interaction between Glu418B and N1′ and show that N4′ is in its iminium form, because of the dimerization of the enzyme, unlike the proposed mechanism showing N4′ as an amino group. Going further in our proposed mechanism and comparing it with steps c−e (Scheme 1) in the common mechanism, we note more significant differences. As previously mentioned, abstraction of the C2 proton by His481B via the N4′ iminium group is not convincing. To agree with the published experimental data, abstraction of the C2 proton has to be achieved by His481B via a relay moiety other than N4′. We turned our attention to H2O688A, which is well positioned in the IRC reactant as a relay for proton transfer between ThDP C2 and Nδ of His481B (Table 2, entries 1b, 3b, and 7b) with a compatible C2−H−O angle (Table 2, entry 3b). In addition, H2O688A is well-stabilized by interactions with several

contained 1842 atoms, including crystallographic water molecules. The HIGH layer (Figure 2, green sticks) contained 97 atoms, including the ThDP cofactor, with no ethyl-pyrophosphate group, the side chains for His481B and His103A, and the carboxylic groups from Asp477B, Glu162A,62,63 and Glu418B. The sequence O Gly116A-Pro117A-N Leu118A, making Hbonds with ThDP N4′H and N3′ was also added along with the crystallographic water molecules superimposed on every TK PDB structure such as H2O880A, H2O883A, H2O688A, H2O882A, and H2O883A, making an H-bond with HN of His69A. This HIGH layer was extended to Glu162A because it is known to play an important role in proton transfers considering the significant decrease in activity upon mutation of this residue.64 Glu418B and Glu162A were set in their neutral form because they directly interact with each other. There were no frozen atoms in this layer. The LOW layer was divided into two parts. The first part containing 237 atoms was free during energy minimization (Figure 2, red sticks), whereas the second (1605 atoms, Figure 2, wire) was frozen. The unfrozen part contained Gly68A, His69A, His103A, Pro104A, Leu118A, Gly119A, Glu162A, Ile191A, His263A, Leu383B, Ile416B, Glu418B, Phe442B, Phe445B, Tyr447B, Asp477B, and ThDP C2H4-O (see the Supporting Information). The atoms included in the HIGH layer and those from the first part of the LOW one were defined iteratively until the result of the geometry optimization came as close as possible to the RX data.

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RESULTS AND DISCUSSION Description of the Structural Model. We focused on the hydrogen bond network in the vicinity of ThDP C2 consistent with crystallographic data (Figure 2). The first optimized geometry at the B3LYP/6-31g level showed a short H-bond (Table 1, entry 1a) reflecting a strong interaction between H2O688A and His481B, together with a lower but still strong interaction with His103A (Table 1, entry 2a) (Figure 3B). In Table 1. Distances between the Targeted Atoms in the Yeast TK Active Site after Geometry Optimization at the B3LYP/ 6-31G Level bond length (Å)

entry

bond type

1

HOH(688A)− Nδ His481B HOH(688A)− Nδ His103A C2−H C2 H− OH2(688A) HO−H(688A) N4′H− OH2(688A) N4′H−C2 N4′−C2 C4′−N4′ H−OGlu418B N1′− HOGlu418B N1′−OGlu418B C2−Nδ His481B

2 3 4 5 6 7 8 9 10 11 12 13

initial model (a)

IRC reactant (b)

transition state (c)

IRC product (d)

1.680

1.679

1.081

1.050

1.704

1.704

2.054

1.475

1.097 1.833

1.097 1.834

1.320 1.263

2.374 0.979

1.016 2.146

1.016 2.145

1.529 1.865

1.670 2.476

2.580 3.276 1.335 1.081 1.454

2.579 3.276 1.335 1.081 1.454

2.532 3.257 1.333 1.103 1.408

2.068 2.966 1.335 1.250 1.208

2.513 4.342

2.513 4.342

2.491 4.449

2.439 4.455 D


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Figure 3. Overview of the model: (A) HIGH ONIOM layer and (B) focus on the interactions among ThDP, H2O688A, and the main residues involved in the ThDP activation mechanism within the HIGH ONIOM layer.

Table 2. Bond Angles between the Targeted Atoms in the Yeast TK Active Site after Geometry Optimization at the B3LYP/6-31G Level bond angle (Å)

entry

angle type

1

HOH(688A)−Nδ His481B HOH(688A)−Nδ His103A C2−H− OH(688A) H−N4′-H N4′−H-C2 N4′-H− OH2(688A) C4′−N4′-H C2-O(688A)− Nδ His481B N4′-O(688A)− Nδ His(481B)

2 3 4 5 6 5 7 8

initial model (a)

IRC reactant (b)

transition state (c)

IRC product (d)

161.22

161.36

154.72

148.93

160.58

160.57

150.31

159.98

154.38

154.20

162.09

130.13

116.20 125.94 153.30

116.19 125.99 153.29

115.54 127.71 161.10

119.81 145.18 127.90

125.64 103.55

125.64 103.57

128.14 121.43

122.29 101.93

70.23

85.58

72.70

70.203

Figure 4. Detailed transition state obtained at the B3LYP/6-31g level. Blue indicates the vector of imaginary frequency (−750.94).

during the ThDP activation process, through a compatible value for C2 stabilization in the IRC product. We note this stabilization process is also visible (Figure 4, blue vectors) in the reaction coordinate, where N4′ moves toward ThDP C2. Our proposed mechanism is consistent with the proton transfer from the H2O688A molecule to Nδ His481B. The corresponding bond length decreased (Table 1, entries 1b−d) to a value compatible for such transfer. We can also assume this proton transfer is assisted by His103A, as illustrated by the strengthening interaction of this residue with the considered water molecule (Table 1, entries 2b and 2d). During the first step in our study, C2H bond stretching is conducted to make no prior assumption about the histidine acceptor (His481B or His103A) of the proton (Figure 3B). The respective distances

residues: His481B, His103A, H2O883A, and of course C2H (Figure 5A). HN4′ also interacts with H2O688A, but the N4′H−OH2688A distance is greater (Table 1, entry 6b) than the ThDP C2H−O688A distance (Table 1, entry 4b). Hence, ThDP C2 deprotonation appears to be much easier when ensured by H2O688A than by N4′, as proven by the marked decrease in C2H−OH2688A bond length. The decrease in both N4′H−C2 and N4′−C2 distances (Table 1, entries 7b−d and 8b−d, respectively) and the unchanged C4′−N4′ distance (Table 1, entries 9b−d) show that the N4′ iminium moiety plays a stabilization role47 on the C2 carbanion or carbene46 (Figure 5B) rather than a base role for C2H abstraction. This is also confirmed by looking at the N4′−H−C2 angle (Table 2, entries 5b and 5d), which increases from the IRC reactant, E


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Figure 5. Intrinsic reaction coordinates (IRC) obtained: (A) IRC reactant and (B) IRC product (in the same orientation).

are very similar [1.679 and 1.704 Å, respectively (Table 1, entries 1b and 2b)]. The energy criterion shows that the water proton moves to His481B. Finally, in terms of bond length and angles, compared with the known mechanism (Scheme 1), the one we propose makes most of the successive steps superfluous. Step a has no reason to exist. Step b is plausible, while the subsequent ones (steps c− e) are combined into one (concerted mechanism), where ThDP activation is not achieved directly by His481B via N4′, but via a water molecule (H2OA688), N4′ playing a stabilization role toward the C2 carbanion or carbene. In addition, our mechanism is consistent with many experimental findings that went unexplained. In particular, it can account for why ThDP activation still occurs with a His481Ala TK variant. In this case, His103A could replace His481B in its deprotonation role of the bridge water molecule HOH688A. The same explanation can be offered for human TK,19 where His481B is replaced by Gln428B (results to be published). Thermodynamic Aspect. The weak gradient around TS displayed in IRC (Figure S1) showed a characteristic profile for enzymatic catalysis. The slight difference between DFT and ONIOM energy [1.9 kJ mol−1 (Table S1)] curves (Figure 6) showed that the ThDP activation pathway built from the model is not modified when the whole protein (RX structure) is considered. In addition, the Gibbs free energy value (at 298.15 K and 1 atm) of the TS (9.33 kJ mol−1) obtained with our ThDP activation pathway in TK is much lower than that obtained by Lie et al. when considering the described pathway without the TK structure (29 kJ mol−1 for the lowest value without thermochemistry corrections, compared with 22.5 kJ mol−1 in our study).40 The Gibbs free energy of the IRC product (−17.19 kJ mol−1) showed the good stabilization of carbanion, even though the “product” remains a reaction intermediate in the overall TK mechanism. These thermodynamic investigations of ThDP activation, considering the TK environment for the first time, support our proposed mechanism. We ran all the calculations again at the DFT B3LYP 6-31g** level. Unfortunately, energy results are inconsistent (Figure S2). The large energy difference between DFT and ONIOM levels [71.66 kJ mol−1 (Table S2)] reveals a

Figure 6. Reaction pathway energy diagram at the 6-31g level: () ONIOM, (---) DFT, and (···) Gibbs free energy.

very strong constraint during optimization of the subsystem of the LOW layer containing the free atoms (Table S3), relative to the RX constraints. From an energy point of view, we can assume that the profile obtained with the protein is characteristic for an enzyme reaction. In addition, it shows a transition state less destabilized than those previously published40,42 and is consistent with the accumulation of a stable carbene observed by Meyer et al.46 Our model also helps to explain why ThDP analogues such as 4′-hydroxy-ThDP65 and 4′-deamino-ThDP34 are not active. We performed the same calculations on both analogues. The theoretical results on 4′-hydroxy-ThDP show that the carbene formed is less stable than the native form (Table S4 and Figure S4). In addition, the Gibbs free energy curve shows that only the IRC reactant exists, not the carbene (Table S4 and Figure S4), the energy of TS being lower than that obtained for the product. With regard to the 4′-deamino-ThDP analogue, we found no TS, leading to the conclusion that activation of ThDP was not structurally feasible with the model described in this work. Finally, we explain why these ThDP analogues are inactive, but with a different justification based on an energy F


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Biochemistry Scheme 2. New Concerted ThDP Activation Mechanism for Yeast TK

but an intrastabilization of the carbene formed. This prompts us to consider different types of activation of the C2 ThDP in other ThDP-dependent enzymes. This new mechanism could be useful for reinterpreting published experimental results and defining the entire TK mechanism more accurately in the presence of both donor and acceptor substrates. Finally, it could offer interesting prospects for studying TK substrate specificity for biocatalytic applications and drug design purposes.

criterion for 4′-hydroxy-ThDP and a structural aspect for 4′deamino-ThDP. Expanding This New ThDP Activation to Some Other ThDP-Dependent Enzymes. More generally, our model may allow a better understanding of some other ThDP-dependent enzyme mechanisms. We examined whether our proposed ThDP activation mechanism, based on TK, can be applied to some other ThDP-dependent enzymes such as the DC (decarboxylase) family and the K1 and K2 (2-ketoacid dehydrogenase) families. A three-dimensional homology study was conducted on DC (Figure S4), K1 (Figure S5), and K2 (Figure S6) structures from the thiamine diphosphate-dependent enzyme engineering database (TEED).66 This consisted of superimposing key ThDP atoms (S1, C2, and N3) in every structure. This revealed the presence of the same constitutive residues of the TK active site (equivalent to His481, His103, and Glu418) and one water molecule (equivalent to HOH688, when this latter was clearly identified in the PDB file) in identical positions relative to those of ThDP.67 Thus, the proposed mechanism for ThDP activation in TK could proceed identically in these ThDP-dependent enzymes.

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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.biochem.5b00787. Energy profile for IRC at the DFT B3LYP/6-31g level, comparison of the geometric parameters at the 6-31G and 6-31G** basis set level, 4′-hydroxy-ThDp reaction pathway energy diagram at the 6-31g basis set level, superimposition between 1TRK and the DC, K1, and K2 families, superimposition of 1TRK_DC−K1K2_fam.pdf, optimized structures as a GAUSSIAN input data file for IRC reactant, transition state, and IRC product at the DFT B3LYP/6-31g level and at the DFT B3LYP/6-31g(d,p) level (these last structures are mentioned but not discussed here), and optimized structures as a GAUSSIAN input data file with the 4′hydroxy-ThDP analogue (PDF)

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CONCLUSION In this study, we propose the first computational model for TK leading to a new, rational activation pathway for ThDP in only one step (Scheme 2). We found a strong interaction between ThDP N1′ and HO Glu418B, but without proton transfer. The crucial point highlighted in this study is that deprotonation of ThDP C2 is not performed by ThDP N4′ as previously claimed, but by His481 involving a water molecule bridge (H2O688A). Thus, ThDP activation proceeds via an intermolecular process in which the role of N4′ is to stabilize the C2 carbanion or carbene. Interestingly, the same residues involved in the new mechanism proposed here could occur in the same positions in some other ThDP-dependent enzymes. The ThDP intrinsic constant seems not to be an intra-activation of the C2,

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest. G


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