Structural and Computational Insight into the Catalytic Mechanism of

Article Cite This: J. Am. Chem. Soc. 2018, 140, 310−318

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Structural and Computational Insight into the Catalytic Mechanism of Limonene Epoxide Hydrolase Mutants in Stereoselective Transformations Zhoutong Sun,†,# Lian Wu,‡,# Marco Bocola,§,∥ H. C. Stephen Chan,⊥ Richard Lonsdale,§,∥ Xu-Dong Kong,‡ Shuguang Yuan,*,⊥ Jiahai Zhou,*,‡ and Manfred T. Reetz*,†,§,∥ †

Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin 300308, China ‡ State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China § Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, Germany ∥ Fachbereich Chemie der Philipps Universität, Hans-Meerwein-Strasse, 35032 Marburg, Germany ⊥ Laboratory of Physical Chemistry of Polymers and Membranes, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH B3 495 (Bâtiment CH) Station 6, CH-1015 Lausanne, Switzerland S Supporting Information *

ABSTRACT: Directed evolution of limonene epoxide hydrolase (LEH), which catalyzes the hydrolytic desymmetrization reactions of cyclopentene oxide and cyclohexene oxide, results in (R,R)- and (S,S)-selective mutants. Their crystal structures combined with extensive theoretical computations shed light on the mechanistic intricacies of this widely used enzyme. From the computed activation energies of various pathways, we discover the underlying stereochemistry for favorable reactions. Surprisingly, some of the most enantioselective mutants that rapidly convert cyclohexene oxide do not catalyze the analogous transformation of the structurally similar cyclopentene oxide, as shown by additional X-ray structures of the variants harboring this slightly smaller substrate. We explain this puzzling observation on the basis of computational calculations which reveal a disrupted alignment between nucleophilic water and cyclopentene oxide due to the pronounced flexibility of the binding pocket. In contrast, in the stereoselective reactions of cyclohexene oxide, reactive conformations are easily reached. The unique combination of structural and computational data allows insight into mechanistic details of this epoxide hydrolase and provides guidance for future protein engineering in reactions of structurally different substrates.

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INTRODUCTION In studies focusing on the elucidation of enzyme mechanisms, stereoselectivity has traditionally served as a unique probe.1 More recently, the rapidly growing area of directed evolution of stereoselective enzymes as practical catalysts in organic chemistry and biotechnology continues to generate a huge set of potentially useful data,2 which in principle can be used to deepen our knowledge of the intricacies of enzyme mechanisms. In endeavors of this kind, crystal structures of enantioselective mutants,3 ideally complemented by kinetic studies and molecular dynamics (MD) computations, are particularly revealing. Such an approach is especially powerful when the directed evolution study includes X-ray structures of both (+)- and (−)-selective mutants which enable access to both enantiomeric products.3a Any notable increase or decrease in activity (in the extreme case complete shutdown) also constitutes a handle for studying mechanisms. © 2017 American Chemical Society

Epoxide hydrolases (EHs) occur widely in many organisms and plants, catalyzing the hydrolysis of epoxides with formation of the corresponding 1,2-diols. The respective biological functions vary according to the organism of origin, which include biosynthesis of natural products, detoxification of toxic epoxides, and cellular signaling.4 Limonene epoxide hydrolases (LEHs) have also been used extensively in organic chemistry for stereoselective synthesis of chiral vicinal diols (and also of epoxides).5 In the present study, we focus on LEH6 and utilize extensive information obtained from stereoselective mutants, kinetics, Xray analyses, and MD computations with the aim of learning more about this widely used enzyme. The hydrolytic desymmetrization reactions of the cyclic epoxides 1 (dubbed Received: September 26, 2017 Published: December 12, 2017 310

DOI: 10.1021/jacs.7b10278 J. Am. Chem. Soc. 2018, 140, 310−318

Article

Journal of the American Chemical Society

as well as quantum mechanical (QM) calculations, the overall results shed new light on mechanistic details of LEH-catalyzed stereoselectivity transformations. The basic mechanistic features derived from an earlier structural investigation6a are shown in Scheme 2, which are supported by a recent QM study.11

CYO1) and 3 (dubbed CYO3), respectively, were used as the model LEH-catalyzed transformations (Scheme 1). Wild-type Scheme 1. LEH-Catalyzed Hydrolytic Desymmetrization of Epoxides 1 and 3

Scheme 2. Proposed Catalytic Mechanism of LEH6a,11

(WT) LEH is a poor catalyst in both reactions (13% ee favoring (R,R)-2, and 4% ee favoring (S,S)-4). These transformations were previously employed as the experimental platform for developing optimal directed evolution strategies which ensure the generation of high-quality mutant libraries with minimal screening effort3a,7 (bottleneck of directed evolution).8 Among them, single code saturation mutagenesis (SCSM),3a based on only one amino acid as building block in saturation mutagenesis at relatively large randomization sites, and triple code saturation mutagenesis (TCSM),7a employing three amino acids, combined with iterative saturation mutagenesis (ISM),9 were systematically explored. TCSM proved to be the most efficient method,7a leading to high-quality mutant libraries which require limited screening.8 Relevant is a recent Rosetta-based study on computationally designed LEH mutants as catalysts in the desymmetrization of cyclopentene oxide (1) and cyclohexene oxide (3), requiring the screening of only 37 variants with the identification of mutants showing moderate to pronounced enantioselectivity.10 Surprisingly, in our study some of the evolved highly stereoselective LEH mutants were found to catalyze the rapid reaction of cyclohexene oxide 3 (CYO3), but not of cyclopentene oxide 1 (CYO1).3a In principle, CYO1 is more strained than CYO3 and can be expected to be hydrolyzed faster. Although the crystal structures of WT LEH and several mutants have been determined, which in some cases help to explain the source of stereoselectivity qualitatively, they are of limited use in revealing the observed drastic switches in substrate specificity.3a As already pointed out, previous directed evolution studies led to moderate to excellent improvements in (R,R)- as well as (S,S)-selectivity for both substrates (Table 1). To understand why some LEH variants are specifically active toward CYO1, whereas others are selective for CYO3, and why stereoselectivity is achieved, we crystallized three LEH mutants (SZ338, SZ348 and SZ529) in complex with CYO1 or its diol product. Coupled with new mutagenesis experiments, molecular docking, MD simulation

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RESULTS AND DISCUSSION Newly Evolved LEH Mutants Improve Enantioselectivity in Reactions of Substrate CYO1. In order to answer the puzzling question why some LEH mutants are completely inactive against CYO1, yet drastically enhancing enantioselectivity in the desymmetrization of CYO3, we screened several mutant libraries previously evolved for CYO3 (Table S1). Several variants were found to be active against CYO1 and had already been identified in the original libraries using CYO3 as substrate, for example, SZ80 (L114 V/I116 V/F139 V). However, their enantioselectivities were found to be poor (66% ee in favor of (S,S)-2, 80% conversion in the case of SZ80). To optimize enantioselectivity, SZ80 was used as a template for iterative saturation mutagenesis (ISM)9 at the 5residue randomization site lining the binding pocket L74/ M78/I80/L103/L147 using SCSM based on phenylalanine as the only building block. Two optimal mutants SZ718 (L74F/ I80F/L114V/I116V/F139V) and SZ719 (L74F/M78F/I80F/ L114V/I116V/F139V) were discovered, showing a selectivity of 92% ee (S,S) and conversion amounting to 56−85%. More importantly, these two variants can also hydrolyze the larger CYO3 with ee-values of 95% and 97%, respectively, yielding (S,S)-4. For comparison purpose, the present and previous results are summarized in Table 1. Crystal Structures and MD Simulations Reveal Flexibility of LEH. In order to throw light on the origin of enhanced LEH enantioselectivity, we determined the crystal structures of mutants SZ529 and SZ348 (Table S2), each bound with inactive substrate CYO1 (pdb: 5YAO, 5YNG). We also resolved the previously reported complex structure of

Table 1. Best LEH Variants As Catalysts in the Hydrolytic Desymmetrization of Substrates 1 and 3 1 ee% variant

sequence

WT SZ92 SZ338 SZ348 SZ529 SZ718 SZ719

L74F/M78F/L103 V/L114 V/I116 V/F139 V/L147 V L74F/M78 V/I80 V/L114F I80Y/L114 V/I116 V M32 V/M78 V/I80 V/L114F L74F/I80F/L114 V/I116 V/F139 V L74F/M78F/I80F/L114 V/I116 V/F139 V

3 c%

ee%

c% source

13(R,R) 72(S,S) − − 10(R,R) 92(S,S) 92(S,S) 311

99 6 − ≤5 8 56 85

4(S,S) 92(S,S) 96(R,R) 99(S,S) 97(R,R) 95(S,S) 97(S,S)

99 99 83 97 99 97 98

SCSM/ISM3a TCSM7a TCSM/ISM7a ISM/this study


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Figure 1. Overall structure and flexibility of LEH. (A) Cartoon structure of LEH. The α-helixes and β-strands were colored by red and blue, respectively. Yellow spheres indicate the location of catalytic site. (B) The sequence secondary structure of LEH. (C) The intrinsic flexibility of LEH calculated from PCA analysis. The cyan vector length correlates with the domain-motion scale. Yellow spheres indicate the location of catalytic site.

Figure 2. X-ray electron density map of LEH variants complex for (A) SZ529, (B) SZ348 ,and (C) SZ338. Black grid: 2Fo-Fc at 1.2σ level. Green grid: Fo-Fc at 3.2σ level. Red grid: Fo-Fc at −3.2σ level. White sticks: side chain of key residues in the catalytic site of LEH. The inactive substrate CYO1 (yellow balls-and-sticks) is in multiple-conformation state in the complex structures of SZ529-CYO1 and SZ348-CYO1 as well as in the resolved complex structures of SZ338-CYO1.

is quite flexible in the binding site, adopting two different conformations which fit both 2Fo-Fc and Fo-Fc maps very well. Interestingly, a conserved water molecule between D132 and Y53 (Figure 2B,C), which is considered to be essential for the catalytic step,6a has been expelled from the catalytic center (Figure 2A). In our previously published crystal structures of SZ338 and SZ348, the Osubstrate-OD101 and Osubstrate-OY53 distances appear too short (2.2−2.4 Å) for a typical hydrogen-bond interaction (2.7−3.2 Å).3a,7a Considering the low occupancy of CYO1 in the asymmetric units of SZ338, we therefore rerefined the crystal structures of SZ338-CYO1 (Figure 2C) introducing multiple orientations for the CYO1 molecule. In all crystal structures, the CYO1 molecules in various orientations fit the electron density maps perfectly in both SZ338-CYO1 and SZ348-CYO1. No close contact is observed for both crystal water and CYO1 molecules in the catalytic regions. Moreover, the highly conserved water molecules in both SZ338-CYO1 and SZ348-CYO1 structures are maintained between D132 and Y53 (Figure 2B,C).

SZ338 with CYO1 as inert “guest” in the binding pocket (pdb: 5YQT)3a by removing all suspicious water molecules and adding multiple ligand conformations. The overall structure of LEH comprises four α-helices and six β-strands, which fold into a compact catalytic pocket (Figure 1). The catalytic trait of LEH is composed of Y53, N55, R99, D101, and D132 (Figure 2). Both Y53 and N55 are located in β1, whereas both R99 and D101 reside in β4, and D132 is in β6. Moreover, we found three flexible regions within the vicinity of the substrate binding pocket, the C-terminal loop (C loop), helix 4 (H4), and loop A. Principal component analysis (PCA)12 on the MD trajectory of Apo WT LEH (pdb: 1NU3)6a indicates that these three motifs can undergo noticeable movements, that change both the shape and volume of the catalytic zone (Figure 1 and Movie S1). This phenomenon was also confirmed by the rootmean-square fluctuation (RMSF) of LEH, which was calculated from the MD simulation trajectory (Figure S1). In the complex structures of SZ529-CYO1 and SZ348CYO1 (Figure 2), we found that the inactive substrate CYO1 312


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Figure 3. Substrate binding modes and protein−ligand interaction fingerprint for (A) SZ529-CYO3, (B) SZ719-CYO1, and (C) SZ719-CYO3. Dash: hydrogen-bond interactions. Yellow balls-and-sticks: substrate molecule. White sticks: side chains of key residues in the catalytic pocket. Protein−ligand IFP are indicated by cyan radar plots. The statistical interaction frequency in the IFP plot was obtained from calculating the protein−ligand interaction frequency in the MD simulations.

Combining the observations from both crystal structures and MD simulations, we conclude that the volume of the inactive LEH variants are too big to stabilize a proper near attack conformation (NAC)13 for the smaller CYO1 substrate prior to reaching the transition state. In such an environment, CYO1 continuously alters its conformations in the catalytic site. However, with a limited space, as in the case of mutant SZ719, both CYO1 and CYO3 are held in active poses, leading to (S,S)-2 and (S,S)-4, respectively. Shedding Further Light on the Active Substrate Binding Mode of LEH. In order to elaborate the atomic details of active substrate−LEH interactions, we performed molecular docking and all-atom MD simulations and protein− ligand interaction fingerprint analysis (IFP) (Figure 3 and Figure S4) for active substrates in complex with LEH, including the complexes SZ338-CYO3, SZ348-CYO3, SZ529-CYO3, SZ719-CYO1, and SZ719-CYO3. In each case, the oxygen atom in the epoxide consistently establishes a strong hydrogen-bond interaction with the protonated D101,6a which is involved in a dedicated hydrogen-bond network with R99, D132, W130, Y53, N55 and a conserved water molecule W1. Among the LEH variants, different mutations in the vicinity of catalytic sites alter the hydrophobic interaction profiles. In SZ338, hydrophobic interactions are found between CYO3 and L35-L103-W130-F134, whereas CYO3 interacts with Y80-L103-V114-F134 in SZ348. In SZ529, identical interactions with L103-I116-F134-F139 are also observed. Both CYO1 and CYO3 are hydrolyzed in SZ719. The IFPs of SZ719-CYO1 and SZ719-CYO3 are similar to each other, both of them interact with F78-F80L103-V116-F134-V139, although the interaction frequency of each residue varies slightly. Interestingly, the number of residues involved in the IFP of SZ719 (7 residues) is much higher than that of SZ338, SZ348, and SZ529 (5 residues).

Furthermore, the orientations of CYO1 in SZ338-CYO1, SZ348-CYO1 and SZ529-CYO1 are different from each other, confirming that CYO1 rotates freely in the binding pocket. Such high flexibility of CYO1 hinders perfect alignment of activated water and substrate in the transition state, as also indicated in the crystal structures, and is responsible for its inactivity toward SZ338, SZ348, and SZ529. To further confirm these assessments, we performed extensive all-atom MD simulations for the complexes SZ338CYO1, SZ338-CYO3, SZ348-CYO1, SZ348-CYO3, SZ529CYO1, SZ529-CYO3, SZ719-CYO1, and SZ719-CYO3. The CYO3 complexes were constructed by molecular docking. It is worth noting that mutants SZ338, SZ348, and SZ529 are either inactive or show extremely low activity toward CYO1. In all cases, CYO1 reorientates itself freely in the trajectories. This is suggested by the higher root-mean-square deviations (RMSD) of both protein and that of CYO1 (Figures S2 and S3, Movies S2−S4). In contrast, SZ719 is active on CYO1, which remains stable in the catalytic zone with a very low RMSD value (Figure S3, Movie S5). Identically, all studied mutants are active on CYO3, which also exhibits a more confined orientation in each case (Figure S3, Movies S6−S9). Interestingly, we also found that the size and shape of the catalytic site in each mutant are noticeably different. The volumes of the catalytic regions are 107 ± 1 Å3, 98 ± 1 Å3, and 102 ± 1 Å3 for mutants SZ338, SZ348, and SZ529 respectively, whereas the volume of CYO1 is only 62 ± 1 Å3. Such noticeable differences are responsible for the CYO1 flexibilities in both crystal structures and all-atom MD simulations (Table S3). However, the volume of SZ719 is much more limited, with a value of 86 ± 1 Å3 which can accommodate CYO1 in a more stable conformation. The computed volume of the CYO3 molecule amounts to ∼75 ± 1 Å3, which results in a much more stable interaction within all mutants. 313


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kcal/mol for SZ179-CYO1 and 14.1 kcal/mol for SZ719CYO3, whereas the longer distance d1 (O2-C1) results in much higher activation energies: 19.3 kcal/mol for SZ179CYO1 and 20.5 kcal/mol for SZ719-CYO3. The catalytic processes and covalent bond changes can be visualized in Movies S10−S12. We extended our QM studies to three other systems: SZ338, SZ348, and SZ529. None of them can hydrolyze CYO1 because of their high flexibilities in the catalytic binding pocket, although SZ529 shows a very low activity (c% = 0.08). Thus, we only simulated these three variants bound with respect to CYO3. Similar to SZ719, SZ348 (Figure S5) also yields (S,S)-4. In this case nucleophilic water W1 favors attack at the C2 (d2 = 3.30 Å) atom in CYO3 with an Ea of 14.3 kcal/ mol, whereas the Ea is as high as 18.6 kcal/mol if W1 attacks C1 (d1 = 4.36 Å). In contrast, W1 in SZ338 (Figure S5) prefers to attack C1 (d1 = 3.28 Å), with an Ea of 12.9 kcal/mol yielding (R,R)-4, whereas the Ea is as high as 16.5 kcal/mol if W1 attacks C2 (d2 = 3.45 Å). Identically, SZ529 also leads to preferential reaction at C1 (d1 = 3.35 Å) with an Ea of 11.0 kca/mol, whereas Ea is as high as 15.7 kcal/ml if W1 attacks C2 (d2 = 3.65 Å). It is well-known that the “angle of attack” (AA) in SN2 reactions is critical for a smooth transformation to occur.14 Therefore, we calculated the AA for both QM (Table S4) and MD simulations (Figure S6). In the present case involving an epoxide, the AA is defined by three atoms (Figure S6A): the oxygen from highly conserved water (Ow), reactive carbon (C1/C2) in the substrate, and the oxygen atom (O1) in the substrate. The AA for each active substrate in the QM calculation is within the range of 145−155°, indicating that Ow-C1/C2-O1 are almost on the same line which is known to be most favorable for a SN2 reactions. This observation is also in a good agreement with another previously reported SN2 enzyme process in a haloalkane dehalogenase process.15 To further confirm our results, we also considered the statistics of our MD simulations for AA (Figure S6). The AA for a favored reaction always shows a value facilitating the SN2 catalytic reaction: SZ338-CYO3 (148 ± 0.1°), SZ348-CYO3 (147 ± 0.6°), SZ529-CYO3 (149 ± 0.1°), SZ719-CYO1 (148 ± 0.1°), and SZ719-CYO3 (150 ± 0.1°). These observations are also in good agreement with previous work.11 The differences in Ea originate from the catalytic pathways with respect to the binding conformation of the substrate (Figure 4 and Movies S10−S12). Specifically, if oxygen atom O2 attacks the distant C1 carbon atom, it undertakes a longer pathway with an unfavorable “angle of attack” (AA). Meanwhile, this oxygen atom would bypass C2 in vicinity and experience additional steric hindrance. In contrast, attacking the neighboring C2 directly results in a better AA with minimal steric effect from C1. Conclusions and Perspectives. In summary, we have applied a stereochemical probe for gaining insight into the substrate stereoselectivity of LEH. Based on crystal structures and mutagenesis, the QM and MD calculations have shed light on the mechanistic intricacies of this enzyme. When substrates approach LEH, they can induce large fluctuation in H4, C loop and loop A, which opens space for the entrance of the respective compounds. Subsequently, an activated highly conserved water molecule, hydrogen-bonded with Y53, N55, and D132, can undergo nucleophilic attack in two different ways: reaction either at C1 or at C2 of the epoxide moiety (Figure 5). If the distance between water and C1 (d1) is

This is mainly because SZ719 has a much smaller catalytic site (volume = 86 ± 1 Å3) than any other variants (volumes = 98− 107 ± 1 Å3). This enables SZ719 to undergo more compact interactions with substrates. Revealing the Source of Stereoselectivity. Quantum mechanics, especially the density functional theory (DFT), is an accurate and efficient method for the study of enzyme mechanisms.14 In order to understand the origin of evolved enantioselectivity, a large active-site model (280−300 atoms) based on crystal structures was designed for a QM study. In this model, we include a highly conserved water molecule, the substrate, and residues R99, W130, Y53, D132, V116, F134, N55, D101, V114, F80, L103, F78, V143, V139 as well as F74 in the corresponding positions of mutant SZ719. The geometry of each catalytic site was submitted to geometry optimization by QM method prior to transition-state scan. Mutant SZ719 is capable of hydrolyzing both CYO1 and CYO3, yielding (S,S)-2 and (S,S)-4 molecules, respectively (Scheme 1 and Figure 4). All LEH variants, in principle, can

Figure 4. Calculated activation energy barrier and substrate−water distances of SZ719. (A) The activation energy barrier of SZ719CYO1. Distance in blue was calculated by QM methods. (B) Distances of C1-O2 and C2-O2 along MD simulations of SZ719CYO1. (C) The activation energy barrier of SZ719-CYO3. Distance in blue was calculated by QM methods. (D) Distances of C1-O2 and C2-O2 along MD simulations of SZ719-CYO3.

generate two different stereoisomers (Schemes 1 and 2). If the highly conserved water molecule W1 (Figure 4) attacks the C2 carbon of the substrate, (S,S)-2 or (S,S)-4 are generated for CYO1 and CYO3, respectively. In contrast, if W1 (Figure 4) attacks the C1 carbon of the substrate, the enantiomers (R,R)2 or (R,R)-4 are generated. We succeeded in gaining insight into the origin of stereoselectivity as follows: In both complexes SZ179-CYO1 and SZ719-CYO3, QM calculations and statistics on the distances of d1 (O2-C1) (corresponding to formation of (R,R)-2) and d2 (O2-C2) (corresponding to formation of (S,S)-4) from MD simulations indicate that d1 is noticeably longer than d2, with statistical p-values d2, LEH will produce (S,S)-products. Hepes (pH 7.0) by the sitting-drop vapor difusion method at 4 °C. The proteins at 20 mg/mL were mixed with the reservoir solution at a ratio of 1:1 in a final volume of 4 μL. The complex crystals of SZ529CYO1 were made by the similar method by soaking the SZ529 crystals in the buffer 1.6 M sodium citrate and 0.1 M Hepes (pH 7.0), together with 10 mM CYO1 for 5−10 min. Diffraction data of mutant SZ529-CYO1 were collected at the wavelength of 0.93911 Å on SSRF beamline 17B of the National Center for Protein Science Shanghai (China). All data collection was performed at 100 K. All data sets were indexed, integrated, and scaled using the HKL2000 package.17 The complex structures of SZ348-CYO1and SZ529-CYO1 were solved by molecular replacement method using the program PHASER18 and the structure of apo-SZ348 LEH (PDB code 5CF2) as a search model. Rounds of automated refinement were performed with PHENIX,19 and the models were extended and rebuilt manually with COOT.20 The structures of SZ348-CYO1 and SZ529-CYO1 complex have been refined to 2.50 and 2.60 Å, respectively. Considering the low occupancy of CYO1 in the asymmetric units of SZ338-CYO1, we re-refined this crystal structure by introducing multiple orientations for CYO1 molecule. The statistics for data collection and crystallographic refinement are summarized in Table S2. Protein and Ligand Structure Preparations. All protein and ligand structures were prepared in Schrodinger Maestro software.21 Protein−Ligand Docking. Docking a ligand to the receptor was performed using Glide.22 Cubic boxes centered on the ligand mass center with a radius 8 Å for all ligands defined the docking binding regions. Flexible ligand docking was executed for all structures. Twenty poses per ligand out of 20,000 were included in the postdocking energy minimization. The best scored pose for the ligand was chosen as the initial structure for MD simulations. Molecular Dynamics Simulations. All unbiased MD simulations were performed in Gromacs 5.1.4.23 All amino acid residues of the protein were modeled according to their protonation states at neutral pH. Residue D101 was found in a protonated state. The protein was centered in a water box with a distance of 11 Å away from the protein. The total number of atoms was approximately 40800:48 Na+ and 39 Cl− ions and about 12,800 water molecules. The Amber99SB*-ILDN force field24 was assigned to the protein, water, and ions, while the ligands were treated by the Amber GAFF2 force field25 through the ACPYPE26 tool. The ligands were submitted to the GAUSSIAN 09 program27 for structure optimization at the Hartree−Fock 6-31G* level prior to the generation of force field parameters. All bond lengths of hydrogen atoms in the system were constrained using M-SHAKE.28 A 10 Å cutoff was used for van der Waals and short-range electrostatic interactions. The whole system was heated linearly at constant volume (NVT ensemble, 1 bar) from 0 to 310 K over 400 ps. Ten ns

shorter than that of C2 (d2), LEH favors the formation of the (R,R)-products. This stereoselectivity is supported by the QMcomputed lower activation energy. We also found that different point mutations of LEH could lead to the rearrangement of the highly conserved water, resulting in (S,S)-products. A relatively compact binding site (e.g., that of SZ719) can stabilize both the smaller substrate CYO1 and the larger substrate CYO3. In contrast, larger binding sites (e.g., those of SZ338, SZ348, and SZ529) destabilize the relatively smaller substrate CYO1 in the near attack conformation (reactive conformation),16 which leads to LEH inactivity toward the smaller substrate CYO1. Our IFP analysis has depicted residues frequently involved in protein−ligand interactions. These residues play an essential role in maintaining a specific substrate conformation which corresponds to a specific product. The X-ray structural results and the insights gained by the theoretical analysis can be expected to be of significant use in future protein engineering of selective LEH mutants.

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MATERIALS AND METHODS

Materials. KOD Hot Start DNA Polymerase was obtained from Novagen. The oligonucleotides were synthesized by Life Technologies. Plasmid preparation kit was ordered from Zymo Research, and PCR purification kit was bought from QIAGEN. The mutant was sequenced in GATC Biotech. All commercial chemicals were purchased from Sigma-Aldrich. Lysozyme and DNase I were purchased from AppliChem. Primers Design, Library Construction, and Screening. Please see our previous work.3a,7a X-ray Structure Determination. The SZ348 variant was crystallized in 2.4 M sodium/potassium phosphate, 0.1 M Tris-HCl (pH 8.5), using the sitting-drop vapor diffusion method at 18 °C. Proteins (at 16 mg/mL) were mixed in a 1:1 ratio with the reservoir solution in a final volume of 4 μL and equilibrated against the reservoir solution. The complex crystals of SZ348-CYO1 were made by soaking the crystals of SZ348 in sodium/potassium phosphate, 0.1 M Tris-HCl (pH 8.5) containing 10 mM of CYO1 and 2.5% (v/v) of acetonitrile as co-solvent for 5−10 min. All crystals were flash-cooled in liquid nitrogen after being dipped into a solution containing 10% glycerol, 2.4 M sodium/potassium phosphate, 0.1 M Tris-HCl (pH 8.5). Diffraction data of mutant SZ348-CYO1 complex were collected at the wavelength of 1.5418 Å on an Raxis IV+2 imaging plate detector. The SZ529 crystals were grown in 1.4 M sodium citrate and 0.1 M 315


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Journal of the American Chemical Society equilibration was performed at constant pressure and temperature (NPT ensemble; 310 K, 1 bar) using the Nose−Hoover coupling scheme with two temperature groups. Long-range electrostatic interactions were computed by particle mesh Ewald (PME) summation. Finally, a 300 ns MD simulation with a time step of 2.0 fs were performed for Apo LEH, SZ338-CYO1, SZ338-CYO3, SZ348-CYO1, SZ348-CYO3, SZ529-CYO1, SZ529-CYO3, SZ719CYO1, and SZ719-CYO3. The MD simulations results were analyzed in Gromacs23 and VMD.29 The binding site volume was calculated by POVME 2.0 software.30 The IFP between protein and ligand was done with PLIP tool.31 More details about IFP can be found in our previous work.32 Figures were prepared in PyMOL and Inkscape.33 The Quantum Mechanics Surface Scan. All calculations were done in GTKDynamo tool,34 a PyMOL interface plugin, using ORCA35 program as the QM engine. QM calculations were performed using the hybrid density function B3LYP. Residues within 5 Å around the substrate, including Y53, W130, N55, D132, F134, I116, R99, F139, D101, L103, V80, L35, and L74 (a total of 280−300 atoms), were included in the QM calculation. The backbones of the residues were restrained with a force constant of 4000 kcal·mol−1·Å2. For each step in the reaction catalyzed by LEH, we performed relaxed potential energy surface (PES) scans, from which we can reconstruct the reaction mechanisms as reported previously.36 All fixed points were located and characterized using an analogous version of the micro/macro iteration algorithm37 with convergence criterion of 0.01 kcal·mol−1·Å−1. The final Hessian was computed to check the existence of a single imaginary frequency (saddle point) or no imaginary frequency (minimum). Steepest descent path calculations were performed for each step so as to verify that the transition states connect the related reactant and product states. Maximum interaction was set to 5000 steps.

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Movie S8: The movements of SZ529-CYO3complex during the MD simulations. Yellow stick: CYO1 molecule. Red sphere: water molecules. CYO1 is relatively stable in the catalytic site (AVI) Movie S9: The movements of SZ719-CYO3complex during the MD simulations. Yellow stick: CYO1 molecule. Red sphere: water molecules. CYO1 is relatively stable in the catalytic site (AVI) Movie S10: The bond forming and breaking in the catalytic process of SZ338-CYO3, calculated by QM methods (AVI) Movie S11: The bond forming and breaking in the catalytic process of SZ719-CYO1, calculated by QM methods (AVI) Movie S12: The bond forming and breaking in the catalytic process of SZ719-CYO3, calculated by QM methods (AVI)

AUTHOR INFORMATION

Corresponding Authors

*[email protected] *[email protected] *[email protected] ORCID

Zhoutong Sun: 0000-0002-9923-0951 Shuguang Yuan: 0000-0001-9858-4742 Manfred T. Reetz: 0000-0001-6819-6116 Author Contributions

ASSOCIATED CONTENT

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S Supporting Information *

These authors contributed equally to this work.

Notes

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b10278. Selectivity data for additional mutants; data collection and refinement statistics for X-ray diffraction experiments; MD simulations and QM calculation data (PDF) Movie S1: The intrinsic flexibility of LEH. Yellow region: H4, green region: loop A, and blue region: C loop (AVI) Movie S2: The movements of SZ338-CYO1complex during the MD simulations. Yellow stick: CYO1 molecule. Red sphere: water molecules. CYO1 is very flexible in the catalytic site (AVI) Movie S3: The movements of SZ348-CYO1complex during the MD simulations. Yellow stick: CYO1 molecule. Red sphere: water molecules. CYO1 is very flexible in the catalytic site (AVI) Movie S4: The movements of SZ529-CYO1complex during the MD simulations. Yellow stick: CYO1 molecule. Red sphere: water molecules. CYO1 is very flexible in the catalytic site (AVI) Movie S5: The movements of SZ719-CYO1complex during the MD simulations. Yellow stick: CYO1 molecule. Red sphere: water molecules. CYO1 is relatively stable in the catalytic site (AVI) Movie S6: The movements of SZ338-CYO3complex during the MD simulations. Yellow stick: CYO1 molecule. Red sphere: water molecules. CYO3 is relatively stable in the catalytic site (AVI) Movie S7: The movements of SZ348-CYO3complex during the MD simulations. Yellow stick: CYO1 molecule. Red sphere: water molecules. CYO1 is relatively stable in the catalytic site (AVI)

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the staff of beamline BL17B of the National Center for Protein Science Shanghai (China) for access and help with the X-ray data collection. M.T.R. thanks the Max-PlanckSociety and the LOEWE Research cluster SynChemBio for generous support. Z.S. thanks the CAS Pioneer Hundred Talent Program (Type C) (reference number 2016-053) for initial start support. J.Z. thanks the Strategic Priority Research Program (B) of the Chinese Academy of Sciences (XDB20000000) and the Science and Technology Commission of Shanghai Municipality (15JC1400403) for fund support. The molecular modelling and molecular dynamics simulation were performed at the Interdisciplinary Centre for Mathematical and Computational Modeling in Warsaw (GB71-3 and GB70-3). We also thank Dr. José Fernando Ruggiero Bachega for helpful advices on QM calculations. We thank Rene de Jong (Groningen/Netherlands) for helpful initial comments.

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