Theoretical Study of Enzyme Promiscuity: Mechanisms of Hydration

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Theoretical Study of Enzyme Promiscuity: Mechanisms of Hydration and Carboxylation Activities of Phenolic Acid Decarboxylase Xiang Sheng, and Fahmi Himo ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b03249 • Publication Date (Web): 24 Jan 2017 Downloaded from http://pubs.acs.org on January 24, 2017

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Theoretical Study of Enzyme Promiscuity: Mechanisms of Hydration and Carboxylation Activities of Phenolic Acid Decarboxylase Xiang Sheng and Fahmi Himo*

Department of Organic Chemistry Arrhenius Laboratory Stockholm University SE-10691 Stockholm Sweden

Corresponding author: [email protected]

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Abstract The cofactor-free phenolic acid decarboxylases (PADs) catalyze the nonoxidative decarboxylation of phenolic acids to their corresponding p-vinyl derivatives. Since these compounds are useful industrially, PADs have potential applications as biocatalysts. Recently, PADs have been reported to also catalyze the hydration and carboxylation of hydroxystyrenes, increasing further their biocatalytic utility. We have used quantum chemical methodology to investigate the detailed mechanisms of both promiscuous reactions. A large model of the active site is designed starting from the crystal structure of PAD from Bacillus subtilis. The calculations suggest new mechanisms, quite different from the literature proposals. For the carboxylation reaction, a carbon dioxide molecule is proposed to be generated from bicarbonate first and then act as the source for the carboxylate group of the product. For the hydration activity, the reaction is suggested to start with the formation of a quinone methide intermediate by protonation of the C=C double bond of the p-vinylphenol substrate. A water molecule then attacks the α-carbon to generate the alcohol product. The enantioselectivity of the hydration reaction is also investigated in this study, and the calculations are able to reproduce and rationalize the observed experimental outcome.

Keywords: reaction mechanism, enantioselectivity, enzyme promiscuity, density functional theory, quantum chemistry, cluster approach


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1. Introduction Phenolic acid decarboxylases (PADs) catalyze the decarboxylation of phenolic acids to their corresponding vinyl phenols (Scheme 1) [1,2]. As important compounds in the cell wall of plants, phenolic acids can be released during the degradation of lignin [3,4]. Since lignin is an abundant and renewable resource and the generated phenolic acid products are useful industrially [5,6], PADs have attracted increasing attention as biocatalysts in recent years [7]. Very recently, PADs have also been found to catalyze two promiscuous reactions, namely the carboxylation [8,9] and hydration [10] of hydroxystyrenes (Scheme 1), extending thereby their potential utility as biocatalysts. Phenolic acids are important in pharmaceutical and industrial applications [11,12], and the PAD-catalyzed carboxylation is therefore an attractive enzymatic method to produce these valuable compounds by fixing CO2. Also, the asymmetric hydration of styrene and its substituted derivatives to produce chiral alcohols is considered to be a challenging reaction in organic chemistry, and an enzymatic protocol for this transformation would be highly valuable.

Scheme 1. The natural (a) and the promiscuous carboxylation (b) and hydration (c) reactions catalyzed by phenolic acid decarboxylases.

We have in a previous study used density functional theory (DFT) calculations and a large model of the active site of PAD to investigate the mechanism of the natural decarboxylation reaction of p-coumaric acid [13]. The calculations gave general support to the previously proposed mechanism involving a quinone methide intermediate [14,15]. However, the substrate was found to be bound in a different orientation compared to previous proposals, namely such that the p-hydroxyl group interacts with the Tyr11 and


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Tyr13 residues (see Scheme 2). This binding mode is similar to what has been observed in the crystal structures of other enzymes with p-hydroxylated aromatic substrates, such as hydroxycinnamoyl-CoA hydratase-lyase (HCHL) [16] and vanillyl alcohol oxidase (VAO) [17]. Very importantly, the Glu64 residue was demonstrated to be the general acid that protonates the substrate. The previously proposed proton source, Tyr19, was instead shown to lower the barrier for the C-C bond cleavage by forming a hydrogen bond with the carboxylate group of the substrate [13].

Scheme 2. Mechanism of the natural decarboxylation reaction of phenolic acid decarboxylase suggested on the basis of previous quantum chemical calculations [13].

Scheme 3. Previously proposed mechanisms of the carboxylation (a) and hydration (b) of hydroxystyrenes catalyzed by phenolic acid decarboxylases [9,10].

In the present paper, we focus on the mechanisms of the promiscuous hydration and carboxylation activities of PAD. The two reactions have been suggested to proceed through a common first step, namely the nucleophilic attack of the β-carbon of the hydroxystyrene substrate on a bicarbonate molecule, resulting in a quinone methide oxyanion intermediate (Scheme 3) [9,10]. In the case of carboxylation, a re-aromatization then takes place to generate the phenolic acid product induced by the elimination of a water molecule with the assistance of a glutamate residue at the active site [9]. For the hydration reaction, a water molecule instead attacks the Cα of the quinone methide 4 Environment ACS Paragon Plus

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intermediate, followed by the collapse of the oxyanion to reform the bicarbonate as a result of the protonation of the β-carbon [10]. Here, we employ the same methodology and active site model as in the previous study [13] to examine the energetic feasibility of these mechanisms. It turns out that already the common first step, the nucleophilic attack of the substrate on the bicarbonate, is associated with a high energy barrier. Instead, alternative mechanisms with much lower barriers are proposed for both the hydration and carboxylation reactions. We also investigate the enantioselectivity of the hydration reaction and discuss its origins. The adopted quantum chemical methodology has previously been successfully used to elucidate a large number of different enzymatic reaction mechanisms [18-22]. In particular, it has in recent years been demonstrated to yield good results for asymmetric enzymatic reactions. [23,24].

2. Computational Methods All the calculations presented here were performed using the Gaussian 09 program [25] with the B3LYP hybrid density functional method [26,27]. Geometries were optimized with the 6-31G(d,p) basis set. Single-point energies at the same level of theory were calculated with SMD solvation model [28] to consider the effects of the enzyme surrounding. In this method, the rest of enzyme, that is not included in the model, is approximated by a polarizable homogeneous medium with some dielectric constant, here set to ε=4. The model does thus not account for the heterogeneity of the enzyme surrounding. Systematic studies have shown that the effect of the solvation decreases quite rapidly with the size of the active site model [29-32]. To obtain more accurate energies, single-point calculations on the optimized structures were performed with the larger basis set 6-311+G(2d,2p). Frequency calculations were performed with the 6-31G(d,p) basis set to obtain zero-point energies (ZPE). Dispersion corrections were obtained using the DFT-D3(BJ) method [33,34]. The energies presented in this paper are thus the large basis set energies corrected for ZPE, solvation and dispersion effects. A table containing the energy breakdown for the stationary points of the proposed mechanisms is provided in the Supporting Information.


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3. Results and Discussion 3.1 Active Site Model Several crystal structures of PAD from various organisms have been obtained [9,3541]. These structures show high overall similarity and the residues in the active sites are highly conserved. Like in our previous study [13], the structure with the Tyr19Ala mutant of PAD from Bacillus subtilis (BsPAD, PDB 4ALB) [40] was chosen to construct an active site model to investigate the reaction mechanisms. The model consists of the pvinylphenol (p-VP) substrate, the bicarbonate, and the following residues that make up the active site pocket: Tyr11, Tyr13, Tyr19, Tyr31, Ile33, Val38, Arg41, Trp62, Glu64, Thr66, Thr68, Val70, Leu72, Ile85, Phe87, Thr98 and Gln102 (Figure 1). Two crystallographic water molecules, Wat1 and Wat2, are also included, as in the previous study [13]. In addition, a third water molecule (Wat3) was also included, as shown in Figure 1. The model consists thus of 312 atoms and has a total charge of −1. Following the results of the previous study on the decarboxylation [13], the substrate was positioned such that the phenolic hydroxyl group points toward the Tyr11 and Tyr13 residues. The hydroxyl group is furthermore assumed to lose its proton upon binding to the active site, as the interaction with Tyr11 and Tyr13 has been shown to acidify the proton considerably [13]. The substrate is therefore modeled in the deprotonated state. Also following the previous study [13], the Glu64 residue that acts as a proton source in the decarboxylation is modeled in its protonated state. Enzyme-substrate complexes with the phenolic hydroxyl forming hydrogen bonds to Arg41 and Glu64 were also considered, but during the optimization of reactant, the phenolate oxygen abstracts a proton from Glu64. This results in the failure to generate a quinone methide intermediate in the following step. Thus, the structures with such a substrate orientation are not productive and will not be discussed further. Since the substrates (p-VP, bicarbonate and Wat3) can bind to the active site in many different ways, a large number of structures of the Michaelis complex (>80) have been optimized in the current study. This was done by preparing starting structures in which the orientations of the substrates relative to each other and relative to the active site residues were varied manually and the geometries were optimized. The structure with the lowest energy among them (called React) is shown in Figure 1 (see Supporting Information for optimized structures and energies of other low-energy complexes). In this structure, Wat3 is found to be in the vicinity of Glu64, and the


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bicarbonate forms multiple hydrogen bonds with the surrounding groups Arg41, Glu64, Wat2 and Wat3. Interestingly, an OH···π interaction is observed between the bicarbonate and the double bond of p-VP.

Figure 1. (a) Schematic illustration of the active site model. (b) The optimized structure of React, which corresponds to the lowest energy among the enzyme-substrate complexes considered in the present study. Atoms marked with asterisks are fixed during the geometry optimization. Distances are given in angstroms. For clarity, only polar hydrogen atoms and the hydrogen atoms on the substrates are shown in the figure.


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3.2 Carboxylation Reaction Mechanism As discussed above, in the previously suggested mechanisms for both the carboxylation and the hydration of hydroxystyrenes, the reaction starts with the nucleophilic attack of the β-carbon of p-VP on the bicarbonate, resulting in a quinone methide intermediate [9,10]. We have optimized a number of transition states for this C-C bond formation reaction starting from different enzyme-substrate complexes. The lowest energy barrier was calculated to be 26.8 kcal/mol relative to React (optimized TS structure is shown in Figure 2 and higher energy TS structures are given in SI). Also the quinone methide intermediate was found to have a very high energy, +22.9 kcal/mol relative to React (Figure 2). These energies clearly show that this mechanism is not energetically viable. It is noticeable that during this step the bicarbonate abstracts a proton from Glu64 in order to avoid a dianionic intermediate. We envisioned therefore that carbonic acid, rather than bicarbonate, could possibly be the carboxylating agent in the reaction. We optimized the corresponding transition state, but it turns out that a similar proton transfer from Glu64 to the carbonic acid still occurs. The calculated barrier for this step is 24.3 kcal/mol, which is comparable to the case with bicarbonate (see SI for optimized structures), and this scenario can also be ruled out.

Figure 2. Optimized structure for the transition state (a) and the quinone methide intermediate (b) in the previously proposed common first step of the carboxylation and hydration reactions. The energies relative to React are given in kcal/mol. For clarity, only a part of the model is shown here. Distances are given in angstroms.

Searching for an alternative mechanism, we envisioned that the carboxylation reaction could start with a proton transfer from Glu64 to the bicarbonate to generate carbon dioxide and a water molecule (Int1C, Scheme 4). The β-carbon of p-VP could then perform a nucleophilic attack on the formed carbon dioxide to give a quinone methide intermediate


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Int2C. The phenolic acid product (ProdC) is finally formed by a proton transfer from the α-carbon of p-VP to Glu64. Thus, the reaction after the generation of the carbon dioxide would be the reverse of the natural decarboxylation reaction catalyzed by PAD shown in Scheme 2.

Scheme 4. Reaction mechanism of the PAD-catalyzed carboxylation of p-vinylphenol suggested on the basis of the present calculations.

Indeed, the calculated energy barriers for this scenario turn out to be feasible. The obtained energy profile is shown in Figure 3 and the optimized geometries of the stationary points are given in Figure 4. The barrier for the CO2 formation (TS1C) is calculated to be 19.5 kcal/mol, constituting the rate-limiting barrier of the reaction. The resulting intermediate (Int1C) is 5.6 kcal/mol higher than React. At TS1C, the breaking O−C bond distance is 1.89 Å and the ∠OCO angle of the forming carbon dioxide is 150°. The Tyr19 residue forms a hydrogen bond with Wat3, which is further hydrogen-bonded to the forming carbon dioxide. In Int1C we note that there is an OH···π interaction between Wat3 and the double bond of the substrate. Here, it should be pointed out that in order to make sure that the lowest-energy 9 Environment ACS Paragon Plus

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barrier is obtained for the rate-limiting first step, we optimized the transition states starting from all enzyme-substrate complexes within 5 kcal/mol from React (11 in total). The optimized TS structures and their relative energies are given in SI.

The following C-C bond formation has a calculated barrier of 8.4 kcal/mol relative to Int1C, i.e. 14.0 kcal/mol relative to React. At the transition state (TS2C), the forming C-C bond distance is 2.31 Å and Wat3 has changed its position to form a hydrogen bond with the forming carboxylate group, creating a linkage between the carboxylate and Tyr19, which is also maintained in the following stationary points. The resulting intermediate Int2C is 2.4 kcal/mol lower in energy than React. From there, a proton transfer from p-VP to Glu64 takes place, with a calculated barrier of 15.7 kcal/mol, completing thereby the reaction mechanism. The enzyme-product complex (ProdC) is calculated to be 10.1 kcal/mol lower than the enzyme-substrate complex (React).

Figure 3. Calculated energy profile for the PAD-catalyzed carboxylation of pvinylphenol.

As discussed above, in all the stationary points, Tyr19 interacts with p-VP or bicarbonate via Wat3 as a bridge (Figure 4). It is interesting that in the decarboxylation reaction, Tyr19 was found to form a hydrogen bond to the carboxylate group of the substrate in the C-C bond cleavage step, and the calculations showed that the barrier was 5.0 kcal/mol higher in the absence of this hydrogen bond [13]. Here, the influence of this residue on the energetics of the carboxylation was also investigated by using a model in


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which Tyr19 was replaced by a phenylalanine residue (see SI). The calculated barriers for all three steps involved in the reaction increased by 3−5 kcal/mol, showing thus that Tyr19 is also important in the carboxylation activity of PAD.

Figure 4. Optimized structures of the stationary points for the PAD-catalyzed carboxylation of p-vinylphenol. For clarity, only a part of the model is shown here. Distances are given in angstroms. 3.3 Hydration Reaction Mechanism Experimentally, it has been shown that both the conversion and enantiomeric excess (ee) of the hydration reaction depend on the concentration of the bicarbonate in the system [10]. Combining this information with the results from molecular docking simulations, the hydration was, as discussed above, proposed to start with the nucleophilic addition of the p-VP substrate to the bicarbonate [10]. However, as shown above, the C-C bond formation


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between the bicarbonate and p-VP is here calculated to be associated with high energies (Figure 2).

Scheme 5. Reaction mechanism of the PAD-catalyzed hydration reaction suggested on the basis of the present calculations.

Instead, on the basis of the calculations, we found a reaction mechanism for the PADcatalyzed hydration that takes place without the formation of a C-C bond, as shown in Scheme 5. The first step of the reaction is the protonation of the Cα-Cβ double bond of pVP. The bicarbonate in this scenario is suggested to act as a proton shuttle between Glu64 and p-VP substrate. A quinone methide intermediate IntH is formed as a result of these two proton-transfer events. The second step is a nucleophilic attack of the water molecule at the α-carbon to generate the alcohol product ProdH. Concertedly, a proton transfer from the water molecule to the bicarbonate takes place, and the proton at the bicarbonate transfers back to Glu64. As shown in Figure 5, this scenario is calculated to be energetically feasible. The optimized geometries of the stationary points in the reaction are given in Figure 6. Similarly to the case of carboxylation, in order to ensure that the lowestenergy stationary points are located, we followed the full reaction paths for the hydration


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reaction starting from the 20 lowest-energy enzyme-substrate complexes (within about 8 kcal/mol from React). This is particularly important for reproducing and analyzing the origins of the enantioselectivity, as will be discussed below.

Figure 5. Calculated energy profile for the PAD-catalyzed hydration of p-vinylphenol.

The first step is calculated to have a barrier of 8.7 kcal/mol, and the resulting quinone methide intermediate IntH is 5.0 kcal/mol lower than React. In the optimized structure of the corresponding transition state TS1H, the proton on the bicarbonate transfers to the βcarbon of p-VP with O-H and C-H distances of 1.17 Å and 1.46 Å, respectively. However, the concerted proton transfer from Glu64 to the bicarbonate is almost completed. Wat3 is found to be in the vicinity of Tyr19 forming hydrogen bonds to the bicarbonate at TS1H, which are also maintained in IntH. The second step is calculated to be rate-limiting with a barrier of 14.4 kcal/mol. At the transition state (TS2H), the distance of the forming C-O bond is 1.98 Å. The experimental barrier of the overall reaction is estimated to be ~15 kcal/mol, as calculated from the rate constant (kcat) of 37 s-1 [10]. Thus, the barrier calculated here is in very good agreement with the experiments.


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Figure 6. Optimized structures of the stationary points for the PAD-catalyzed hydration of p-vinylphenol. For clarity, only a part of the model is shown here. Distances are given in angstroms.

We have also considered a number of alternative scenarios for the substrate protonation step (React → IntH), all of which turned out to have significantly higher energies compared to the mechanism described above (Scheme 5). In the first, we examined whether Glu64 could protonate the substrate directly, i.e. without the mediation of the bicarbonate. However, the barrier was found to be as high as 22.4 kcal/mol, i.e. ca. 14 kcal/mol higher than TS1H. Another way is to have the Wat3 molecule as an extra mediator of the proton transfer between the bicarbonate and the substrate. Also this option was found to have a high barrier, 19.0 kcal/mol relative to React. Finally, we also tested the case in which Glu64 is in the deprotonated form, meaning that the bicarbonate is the proton source. The energy for the quinone methide intermediate was calculated to be 15.0 kcal/mol higher than the corresponding reactant, which is very high considering that IntH is at -5.0 relative to React. The optimized geometries of these alternative scenarios are given in the Supporting Information. We also investigated the case in which the bicarbonate is not included in active site model at all. Very interestingly, in the absence of bicarbonate, a water molecule takes its place as a proton shuttle, bridging between Glu64 and the substrate (see SI for optimized geometries and calculated energy profile). The calculated barrier for the first step is 13.0


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kcal/mol, i.e. 4.3 kcal/mol higher than with the bicarbonate. However, the barrier for the second step, the rate-limiting nucleophilic attack of the water molecule, increases only by 0.7 kcal/mol (to 15.1 kcal/mol). The calculations indicate thus that the reaction could still occur without the involvement of the bicarbonate, albeit with a lower rate. This is consistent with the experimental findings that the reaction still has a conversion (more than 40%) in the absence of bicarbonate [10].

3.4 Enantioselectivity of Hydration Reaction Turning to the enantioselectivity of the hydration reaction, it was experimentally shown that the alcohol product with S-configuration is produced in up to 87 % ee [10]. This corresponds to a transition state energy difference of 1.6 kcal/mol. The enantioselectivity is dictated at the second step, the nucleophilic attack of the water molecule on the quinone methide intermediate. As mentioned above, since the substrates of the promiscuous hydration reaction (p-VP, water and bicarbonate) are smaller than the natural phenolic acid substrate, they can be in many different orientations relative to each other in the active site pocket. As discussed above, we have followed the full reaction paths for the 20 lowest energy enzyme substrate complexes. The optimized structures of the nucleophilic attack transition states and the calculated energy barriers are given in the SI. The lowest energy transition state located for the selectivity-determining step corresponds indeed to the formation of the S-enantiomer of the product (TS2H in Figure 6), reproducing thus the experimental outcome. The calculated barrier for the lowest energy transition state leading to the R-product (TS2H', Figure 7) is calculated to be 11.7 kcal/mol, which is 2.3 kcal/mol higher than that for the S-product. The calculated energy difference is thus in very good agreement with the experimental results [10]. Analysis of the transition state geometries can give some hints to the factors controlling the selectivity. We first note that the quinone methide intermediate is anchored in the active site by the hydrogen bonding interaction of its oxygen with the two tyrosines, Tyr11 and Tyr13. It can moreover have either the Si or Re faces of its double bond facing the Tyr19 side of the active site. The bicarbonate, on the other hand, is positioned in the other end of the active site by hydrogen bonds with residues Arg41, Glu64 or Thr66. The position of the nucleophilic water (Wat3) is more flexible, as it can be either above the substrate, in the vicinity of Tyr19 (see Figure 7), or below the substrate, close to Val70. It turns out that in the lowest-energy transition states, leading to both the S- and the Rproducts, the water molecule is favorably located in the former position, forming a 15 Environment ACS Paragon Plus

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hydrogen bond to the Tyr19 (and also to the bicarbonate to which it delivers a proton at the TS). The main difference between the transition states is then which face of the quinone methide intermediate is directed to the nucleophilic water. That is, how the newly-formed methyl group of the quinone methide is pointing. In the TS leading to the R-product (TS2H'), the methyl group points toward the Tyr31 side chain, experiencing a steric clash, while in the TS leading to the S-product (TS2H), the methyl points toward the area defined by Val70 and Ile85, which is less crowded (see Figure 7). This difference seems to govern the enantioselection of the nucleophilic attack.

Figure 7. Optimized structures and schematic drawings of the selectivity-determining transition states leading to S-product (a) or R-product (b). The energies relative to React are given in kcal/mol. Distances are given in angstroms.

Finally, we have also considered the enantioselectivity in the absence of bicarbonate. Experimentally, the product is found to form as a near racemate [10]. In our model calculations without bicarbonate, the structure of the nucleophilic attack TS is somewhat different compared to the case with bicarbonate (TS2H), the most important difference being that the nucleophilic water molecule now is located below the substrate, because it has to deliver the proton directly to Gu64 (see optimized structures in SI). The energy difference between the most favorable pathways leading to the S- or R-products is now


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calculated to be 1.2 kcal/mol. This value is slightly overestimated compared to the experimental outcome (near racemate). Nevertheless, the result is quite satisfactory, since the trend in enantioselectivity is well reproduced. Namely, the calculated difference in energy in the absence of bicarbonate is calculated to be smaller than the case with bicarbonate (1.2 vs. 2.3 kcal/mol).

4. Conclusions We have in the present paper employed DFT calculations to investigate the detailed mechanisms of two promiscuous reactions of phenolic acid decarboxylase, namely the hydration and carboxylation of hydroxystyrenes. A large model of the active site (>300 atoms) was designed starting from the crystal structure of PAD from Bacillus subtilis. Considering that the substrates of the promiscuous reactions (p-vinylphenol, bicarbonate and water) are small than the natural phenolic acid substrate, a large number of enzymessubstrate complexes were optimized to find the lowest-energy binding mode. The previously-proposed common first step of the hydration and carboxylation mechanisms, the nucleophilic attack of the β-carbon of vinylphenol substrate on the bicarbonate was first ruled out on the basis of its high energy barrier. Instead, the current calculations suggest new mechanisms that have feasible energy profiles. For the carboxylation reaction, it is suggested carbon dioxide is first generated from bicarbonate by a proton transfer from Glu64. This step is calculated to be ratedetermining for the reactions. The β-carbon of p-VP then performs a nucleophilic attack on the formed carbon dioxide to give a quinone methide intermediate, which next transfers a proton back to Glu64 to yield the final phenolic acid product. For the hydration mechanism, the reaction is proposed to start with the formation of a quinone methide intermediate by protonation of the C=C double bond of the pvinylphenol substrate. Glu64 is suggested to be the general acid responsible for this step, while the bicarbonate acts as a proton shuttle, lowering the barrier for the proton transfer. A water molecule then performs a nucleophilic attack at the α-carbon of the quinone methide, concertedly with a proton transfer from the water to Glu64, over the bicarbonate shuttle, to generate the alcohol product. The water addition is calculated to be rate-limiting step with a barrier in good agreement with the experimental data. The calculations confirm, furthermore, that the hydration reaction can take place using the same mechanism also in the absence of the bicarbonate, albeit with a higher barrier.


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The enantioselectivity of the hydration reaction was also investigated in this current study. The calculations could reproduce the experimental outcome, the preferred formation of the S-alcohol, and also provide a rationale for this observation. The mechanistic insight provided from the current calculations will be valuable for the design of PAD variants with wider substrate scope and better selectivity properties.

Supporting Information Optimized geometries and energies of different enzyme-substrate complexes and of the alternative mechanisms mentioned in the text. Breakdown of energies and Cartesian coordinates of the stationary points of the proposed mechanisms.

Acknowledgment We thank Prof. Kurt Faber for valuable discussions. Financial support from the Swedish Research Council, the Göran Gustafsson Foundation, and the Knut and Alice Wallenberg Foundation is acknowledged.

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Table of Contents Graphic


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Figure 1 155x202mm (300 x 300 DPI)

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Figure 2 47x15mm (300 x 300 DPI)


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Figure 4 135x125mm (300 x 300 DPI)


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Scheme 1 60x26mm (300 x 300 DPI)


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Scheme 4 114x91mm (300 x 300 DPI)


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Scheme 5 107x79mm (300 x 300 DPI)


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Graphical Abstract 20x4mm (300 x 300 DPI)


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Theoretical Study of Enzyme Promiscuity: Mechanisms of Hydration

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