MM Molecular Dynamics Investigations of the Substrate Binding

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QM/MM Molecular Dynamics Investigations of the Substrate Binding of Leucotriene A4 Hydrolase: Implication for the Catalytic Mechanism Xia Mu, and Dingguo Xu J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b04203 • Publication Date (Web): 02 Jul 2018 Downloaded from http://pubs.acs.org on July 3, 2018

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The Journal of Physical Chemistry

QM/MM Molecular Dynamics Investigations of the Substrate Binding of Leucotriene A4 Hydrolase: Implication for the Catalytic Mechanism Xia Mu and Dingguo Xu* MOE Key Laboratory of Green Chemistry and Technology, College of Chemistry, Sichuan University, Chengdu, Sichuan, P. R. China 610064

* To whom correspondence should be addressed: [email protected] (D.X), Tel: 86-28-85406156

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Abstract LTA4H is a monozinc bi-functional enzyme which exhibits both aminopeptidase and epoxide hydrolase activities. Its dual functions in anti- and pro-inflammatory role have attracted widely attention of the inhibitor design. In this work, we tried to construct Michaelis complexes of LTA4H with both native peptide substrate and LTA4 molecule using combined quantum mechanics and molecular mechanics molecular dynamics simulations. First of all, the zinc ion is coordinated by H295, H299 and E318. For its aminopeptidase activity, similar with conventional peptidases, the fourth ligand to zinc ion is suggested to be an active site water, which is further hydrogen boned with a downstream glutamic acid, E296. For the epoxide hydrolase activity, the fourth ligand to zinc ion is found to be epoxy oxygen atom. The potential of mean force calculation indicates about 8.5 kcal/mol activation barrier height for the ring-opening reaction, which will generate a metastable carbenium intermediate. Subsequent frontier molecular orbital analyses suggest that the next step would be the nucleophilic attacking reaction at C12 atom by a water molecule activated by D375. Our simulations also analyzed functions of several important residues like R563, K565, E271, Y383 and Y378 in the binding of peptide and LTA4.

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1. Introduction The leukotriene A4 hydrolase (LTA4H) is a bi-functional zinc containing enzyme with epoxide hydrolase and aminopeptidase activities.1,

2, 3, 4

Particularly, it can

catalyze the conversion of leukotriene A4 (LTA4) to leukotriene B4 (LTB4), a potent inflammatory activator and neutrophil inducer, via epoxide hydrolase mechanism.3 Different pathological conditions and diseases,5 e.g., connective tissue disease and sepsis, have been found to be connected with overproduction of LTB4. Its peptidase activity specifically acts on the N-terminus of peptides, esp., Arg at the N terminus, and its role in the cigarette smoke induced emphysema has also been identified recently.6 Quite interestingly, two activities share the same substrate binding pocket, but the reason to the mechanistic difference remains unknown. In past decades, there were numerous X-ray structures resolved.7, 8, 9, 10, 11, 12, 13, 14, 15

Majority efforts have been applied to understand the mechanism of epoxide

hydrolase due to its important physiological contributions. On the other hand, its aminopeptidase mechanism seems not to be unique, and the extensive discussion has been reported on this kind of mechanism.4 Basically, both mechanisms share one common active site, e.g., the zinc ion is coordinated by H295, H299 and E318. Major debates around the fourth ligand candidate to Zinc ion cause different activities of epoxide hydrolase and aminopeptidase. Traditionally, for the aminopeptidases like carboxypeptidase (CPA),16, thermolysin (TLN),20,

21

17

angiotensin converting enzymes (ACE)18,

19

and

nearly a conserved HEXXH zinc binding motif and a

downstream E residue can be identified for those peptidases. The fourth ligand to zinc ion is a water molecule, which is further hydrogen bonded with an active site glutamic acid residue. This Glu residue will acts as general acid/general base in the subsequent hydrolysis reaction. Not surprisingly, one glutamic acid residue, E296, can be located close to the zinc binding motif, which is considered to take the same role as peptidase does. A conventional general acid/general base (GAGB) mechanism22, 23, 24 for LTA4H as a peptidase can be summarized in Scheme 1. For the mechanism of epoxide hydrolase, two major proposals related to binding models have been reported. The putative interactions between enzyme and LTA4 are 3



given in Scheme 2. Some common features for two pathways can be also located, e.g., salt bridge formed between the carboxyl group of LTA4 and the guanidino group of R563, and the backbone of C7-C20 fatty acid part deeply embedding into a L-shaped narrow hydrophobic binding pocket as shown in Figure 1. Site-specific mutagenesis and kinetic study have confirmed the E271 plays critical role in epoxide hydrolase mechanism.8 Meanwhile, the requirement of one water molecule in the initial step for the ring opening has also been suggested.3, 4, 11 The mechanistic proposal via the epoxide hydrolase is shown in Scheme 2A, in which the fourth ligand of zinc ion is a water molecule. At the same time, the epoxy atom is stabilized by a hydrogen bond with the Y383. This model suggests an acid-induced activation of epoxide ring-opening mechanism, in which the hydrogen atom of the polarized water molecule attacks the epoxy atom with a SN1 reaction leading the bond broken between C6 and oxygen. However, this mechanism cannot well answer the question that E271 directly participates in the conversion of LTA4 to LTB4. The second model is the epoxy atom acted as the fourth ligand of zinc atom.25 Initially, the E271 was assumed to be in its ionic state. This mechanism clearly implies that the proton migration from water to epoxy oxygen atom. However, it is hard to believe that the OH bond of water can break when it is hydrogen bonded with a deprotonated carboxylate group and not be activated by zinc ion. As shown in Scheme 2B, the zinc ion could act as a Lewis acid catalyst to activate the epoxy atom with double-hydrogen atom migration to break the epoxide ring. Subsequently, the production of LTB4 involves charge delocalization over the conjugated triene system (C6-C12).9, 26 Some important residues like D3759 and Y37826,

27, 28, 29

have been well studied in the functions of catalysis and

enantioselectivity. In this work, we will just focus on the substrate binding modes and the first step of ring opening of epoxide ring as an epoxide hydrolase. Recently, the inhibitor design for LTA4H has received much attention due to its dual functions in anti- and pro-inflammatory role.14, 25, 30, 31, 32, 33, 34 However, most of inhibitors show no inhibition specificity against activities of both peptidase and epoxide hydrolase, except the recent reported ARM1,14 which was shown to bind in the hydrophobic pocket but leave the peptidase active site unoccupied. Although 4


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kinetic investigations and crystal structures have been reported for both aminopeptidase and epoxide hydrolase to some details, some important issues are still not well understood. In particular, reasons to bifunctional mechanisms are still under shadow. Recently, the X-ray structures for E271A, D375A and R563A reported by Stsiapanava et al15 clearly show that the fourth ligand to zinc ion should be the epoxide oxygen for the epoxide hydrolase mechanism. However, detailed substrate binding modes for different substrates (peptides or LTA4) in wild type (WT) enzyme is still highly desired for future inhibitor design. Theoretical simulation from microscopic angle represents alternative way to address these issues. In this work, intensive molecular dynamics (MD) simulations will be carried out to identify the recognition differences between aminopeptidase and epoxide hydrolase. 2. Computational Details For bond breaking and formation occurring in enzyme active site, multi-scale method like the combined quantum mechanics and molecular mechanics (QM/MM) method24, 35, 36, 37, 38 has shown its power to tackle these issues. In present study, the self-consistent-charge density functional tight binding (SCC-DFTB) theory39, 40, 41 was employed to describe the electronic structure calculation for atoms in QM region. Because specifically parameterization has been done for biological zinc ion,42 simulations using SCC-DFTB associated with force field have been successfully carried out for several important zinc-containing enzymes.17, 43, 44, 45 CHARMM all atom force field was applied for all environment atoms. All calculations reported here were carried out using CHARMM with a SCC-DFTB interface.46 2.1 Molecular Dynamics Simulations As we have mentioned above, in this work, we will investigate two distinct enzyme-substrate complexes (ES), which exhibit aminopeptidase and epoxide hydrolase mechanisms, respectively. Therefore, two different substrate binding structures will be constructed for two activities. Putative interactions between substrates and enzyme are depicted in Figure 2. Atom name for protein follows the CHARMM convention. Generally, two activities share the same active site with the zinc ion coordinated with H295, H299 and E318. The identity of the fourth ligand to 5



zinc ion is the key to understand the substrate binding modes and mechanisms. Like other peptidases, we simply simulate the fourth ligand to zinc ion as a water molecule. This water is further hydrogen bonded with a downstream glutamic acid residue, E296. The initial structure (as a peptidase) was extracted from protein data bank (PDB entry code 3B7S),12 which is E296Q complexed with a tripeptide substrate of R1'-S2'-R3'. We then manually convert Q296 to E296 to recover its wild type (WT) form. The protonation states for all titratable residues are carefully assigned based on their nearby environment with pH=7, and further confirmed by PROPKA tool.47 In current simulation for peptidase activity, the QM region consists of the zinc ion, the putative active site water, the side chains of the protein ligands (H295, H299 and E318), side chain of E296 and the entire substrate. Total number of atoms in QM region is calculated to be 110. On the basis of Scheme 2, two possible mechanisms for epoxide hydrolase activity have been proposed. The difference can be simply located at the fourth ligand to zinc ion. In this case, the initial structure was adopted from the X-ray structure (PDB id 2VJ8),10 which is a crystallography structure of WT enzyme complexed with a hydroxamic acid inhibitor. Due to close topology of hydroxamic acid and LTA4, this structure can be used to recover the initial model for epoxidase in molecular dynamics (MD) simulations. For the fourth ligand to zinc ion, we built two models. The first model with epoxide hydrolase activity is one water molecule coordinated with Zn, which is postulated to be hydrogen boned with E271. The second model is the epoxy oxygen atom is simulated as the fourth ligand to zinc ion. As shown by Stsiapanava et al.,15 one water molecule should be nearby, which also forms hydrogen bond with E271. For the protonation status of E271, we simply assigned it to be protonated according to the Scheme 2B. For the epoxide hydrolase activity, the QM region then consists of the zinc ion, two putative active site water molecules, the side chains of the protein ligands (H295, H299 and E318), side chains of E271 and D375, and the entire LTA4 substrate. Totally 111 atoms are included in QM region. All constructed models were subjected to be solvated in a pre-equilibrated TIP3P48 water sphere with a radius of 30 Å centered at the Zinc ion, followed by 30 6


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ps solvent molecular dynamics (MD) with all protein and substrate molecules fixed to obtain uniform solvation. Stochastic boundary condition49 was applied to save computational cost. The covalent interface between the QM and MM regions was described using hydrogen link-atom scheme.50 The group-based switching approach51 was applied to treat the non-bonded interactions throughout the simulations. SHAKE algorithm52 was also applied to maintain all hydrogen atoms involved in covalent bonds. For each system, the temperature was slowly heated to 300K within 50 ps, followed by additional 1 ns equilibration. Subsequent 9 ns QM/MM MD simulation was carried out for data analysis. 2.2 Reaction Mechanism Simulation Since the peptidase mechanism has been extensively investigated, we will just focus on the epoxide hydrolase mechanism, and only for the first step of epoxide ring opening. The reaction coordinate was defined as the distance of C6-OB bond with a range of [1.45, 2.25] Å. First of all, the reaction coordinate driving method53 was applied to map the minimum energy path (MEP) for this ring opening process. To include the protein environment entropy contribution, we then calculated the potential of mean force (PMF) for the reaction. All MEP geometries along the reaction coordinate were applied as the initial structure for PMF calculations. To enhance the sampling, the umbrella sampling approach was employed by adding a biasing potential to its force field. The harmonic constraints were set around 100-300 kcal/(mol·Å2). Total of 9 windows were used in PMF calculations, in which 100 ps constrained MD simulation was applied with first 60 ps for heating to 300 K and equilibration and last 40 ps trajectory for data analysis. In particular, the constraint MD calculations were performed with a 1 fs time step and the SHAKE algorithm for covalent bonds involving non-transferring H atoms. Finally, weighted histogram analysis method (WHAM) was used to obtain the PMF.54, 55 3 Results and Discussion 3.1 Michaelis Complex for Aminopeptidase In this work, a tripeptide substrate of R1'-S2'-R3' (primes denote they are residues of polypeptide substrate) is simulated in the active site to examine the peptidase 7



activity of LTA4H. Total of 10 ns QM/MM MD simulation was carried out to explore the detailed substrate binding information. Throughout the simulation, the whole system is maintained very well, which is evidenced by the root mean square deviation (RMSD) of 1.90 ± 0.10 Å (Figure 3A) for the protein backbone atoms compared to X-ray structure. One snapshot randomly recorded from the MD trajectory was depicted in Figure 4A, and some selected statistical average bond distances and angles were summarized in Table 1. First of all, the tetra-coordination status for Zn is kept steadily along the MD simulation. For example, the ligand bonds between protein and zinc ion are calculated to be of 2.00 ± 0.06Å for Zn-Nε2(H295), 2.02±0.06Å for Zn-Nε2(H299) and 2.06 ± 0.07Å for Zn-Oε1(E318), which agrees with X-ray structure very well according to Table 1. Meanwhile, the fourth ligand is simulated to be a water molecule with ⋯( ) = 2.07 ± 0.06 Å. This water molecule is further hydrogen bonded with E296 with ⋯ ( ) =2.25 ± 0.36 Å. In fact, such stable hydrogen bond could strongly support that E296 can serve as the general base to abstract one proton from this water molecule. Previous work of E296Q12 totally inactivated the hydrolysis of R1'-S2'-R3', which further confirms E296 must have some critical functional roles in the catalysis. From the Figure 2A, the whole recognition sites for the peptide substrate are fully occupied by polar residues, which can provide the special binding environment. At the subsite of R3', two positively charged residues, R563 and K565, are identified as two terminal residues, which are suggested to regulate the length of the peptide according to crystal structure.12 Indeed, for the binding of R1'-S2'-R3', its C-terminal carboxylate group is mainly recognized by R563 with salt bridge, evidenced by the distance of 1.69 ± 0.10 Å and 1.75 ± 0.11 Å for OT1-Hη22(R563) and OT2-H(R563), respectively. Moreover, another positively charged residue of K565 is also located nearby. However, K565 does not show any strong connections with the substrate due to relatively long distance away from the substrate. As we can see from the mutagenesis studies of R563A and K565A reported by Rudberg et al.11, the former 8


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one significantly decreases the binding affinity of peptide substrate, while the latter one has no such effect. Our simulations then agree with experimental data very well. Nevertheless, the existence of R563 and K565 could largely restrict the length of the peptide residue. Indeed, LTA4H prefers tripeptide substrate. For the N-terminal amino group on R1′, significant hydrogen bond network is formed with Q136, E271 and E318. Furthermore, there are two guanidino groups for the substrate, which have different recognition environment. At the R1′ site, the guanidine group can be stabilized by D375 with a weak hydrogen bond according to Table 1 and several bulky solvent water molecules. The guanidino group on R3′ is then fully exposed to solvent water molecules. On the other hand, the E271 is unique compared with other peptidases. E271 here only constitutes an N-terminal -NH3 group recognition site but has no functions in the catalysis, as suggested by Rudberg et al.8 We have to emphasize here that the hydrogen bond network around the scissile amide carbonyl oxygen atom is one of the key issues during the hydrolysis of peptide, which has been addressed to some details in the study of other peptidases like ACE, TLN and CPA. For ACE and CPA, conserved tyrosine residue can be identified (Y501 for ACE and Y248 for CPA), while a histidine residue of H231 for TLN can be found, which could provide additional stabilization effects to both the ES complex and the tetrahedral intermediate. Similarly, Y383 in LTA4H can have the same function, evidenced by 1.69 ± 0.10 Å for ⋯

(). To further illuminate the structure similarity between these enzymes, we then plotted an overlap of LTA4H, ACE and TLN in Figure 4B. Unfortunately, due to inappropriate orientation of ligand residues to Zn in CPA, we then included it in the Figure 4C separately. However, it is well known that ACE and CPA have a very close substrate binding pocket.24 Of course, some differences between LTA4H and other peptidases can also be found, e.g., chloride ion dependent activity of ACE. As we have discussed a lot before,24 for the zinc containing aminopeptidases, GAGB mechanism seems to be a natural choice. Indeed, due to nearly identical zinc binding motif and substrate recognition environment among peptidases as presented 9



above, we then believe that the hydrolysis of a peptide substrate catalyzed by LTA4H should be also subjected to GAGB mechanism summarized in Scheme 1. Therefore, we will not further carry out corresponding reaction mechanism simulations here. 3.2 Epoxide Hydrolase Mechanism First, efforts to find a stable complex structure according to Scheme 2A finally failed. We did see that the water can stably form a ligand bond with Zn. However, it could not form hydrogen bond with E271, but with E296 instead. Such kind of model cannot provide any supporting evidences that E271 is directly involved in the reaction, as suggested by kinetic investigations8. Of course, no direct experimental data tell us whether or not E296 directly participates in the reaction with epoxide hydrolase activity. It has been well accepted that E296 plays the critical role during the hydrolysis of a peptide substrate.56, 57 Therefore, we can simply conclude that the Scheme 2A might not be a favorable pathway for the activity of epoxide hydrolase. We will then focus on the second model (Scheme 2B) for the epoxide hydrolase, in which the epoxy oxygen atom is the fourth ligand to zinc ion. In fact, a recent structural study15 on mutated LTA4H complexed with LTA4 clearly shows that the epoxy oxygen atom should be directly coordinated with Zinc ion. Another important issue has to be addressed is the protonation status of E271. Since the complete epoxide hydrolysis reaction involves several protons migration processes, the active site water molecule should be activated by a Lewis acid catalyst. On the other hand, the zinc ion has already acted as an acid catalyst to activate epoxy atom. Therefore, the only candidate to polarize the water molecule would be E271 on account of the E271A completely inactivating the enzyme.8 To this end, E271 in this work is approximated to be in its protonated status when the LTA4H functions as the epoxide hydrolase. Total of 10 ns QM/MM MD simulation was carried out to examine the binding pattern of LTA4 bound within the active site. Overall, the RMSD of 1.76 ± 0.21 Å (Figure 3B) indicates the enzyme was maintained very well along the simulation. To further illustrate interactions between LTA4 and enzyme environment, one snapshot randomly extracted from MD trajectory is displayed in Figure 5. Some selected 10


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geometric parameters are summarized in Table 2. The Zinc ion keeps its tetra-coordination with three ligands from enzyme environment, i.e., H295, H299 and E318, judged by ( ) = 1.98 ± 0.05Å, ( ) = 1.98 ± 0.05Å and ( ) = 2.22 ± 0.40 Å, respectively. The fourth ligand to zinc ion is shown to be the epoxy oxygen atom of OB with a ligand bond distance of 2.27 ± 0.24 Å. This kind of binding structure means that the epoxy ring has the possibility to be activated by zinc ion. The Zinc ion then has the function of Lewis acid catalyst. Another important issue is whether or not the first step of ring opening reaction involves the water molecule, which is supposed to be hydrogen bonded with E271. However, such a water molecule cannot stably stay close to epoxy ring according to our MD simulation. According to Figure S1 and Table 2, pretty large fluctuation can be found for the distance of =6.47 ± 2.63 Å. Therefore, we can safely suggest the catalysis of ring opening should not require participation of the water molecule as well as the E271. This will further be examined by subsequent free energy simulation and corresponding frontier orbital analyses. For the stabilization of LTA4, significant hydrogen bond network formed with enzymes can be observed. For example, the epoxy oxygen atom is stabilized by phenolhydroxyl group of Y383 with () = 2.44 ± 0.48 Å. Similar with the binding of the tripeptide molecule, the terminal carboxylate group is also recognized by R563 via a stable salt bridge, evidenced by the statistically averaged distances of 1.85 ± 0.31 Å for the

!("#)

and 1.92 ± 0.40 Å for

$("#) .

As shown in Figure 1, a long and narrow L-shaped pipe-like tube is surrounded by residues like Q136, A137, W311, P374, D375, A377, W378 and F314. While at the bottom of the pipe, some residues like P382, F362, V367, L369 and W315 can be also identified. Such geometrical pattern can largely restrict the substrate movements and potent inhibitor design, esp., for enzyme like LTA4H. In fact, a long C13-C20 fatty acid tail is deeply embedded into an L-shaped binding pocket. Some interesting features can be easily found based on our simulation. According to Table 3 and Figure 11



5, a coplanar structure for the triene system of C7-C12 can be observed, evidenced by averaged dihedral angles of φ0 = -11.5 ± 22.6°, φ1 = 174.4 ± 4.3°, φ2 = -170.8 ± 7.0° ,

φ3 = -174.2 ± 4.4°, φ4 = 170.6 ± 7.7°, φ5 = 1.4 ± 9.0°. The definition of all six dihedral angles follows the Ref.26. All these dihedral angles indicate that conformation of LTA4 can be maintained during the dynamics simulation, which could finally yield LTB4 rather than other isomers. Clearly, narrow binding pocket of LTA4H could largely restrict bond rotations of the triene systems. Experimentally, a nearby tyrosine residue, Y378, has been suggested to serve the function to regulate the enantiomer for the final product. Therefore, it would be interesting to further examine interaction between C7-C12 triene framework and Y378. In order to clarify the interactions between two conjugate systems, we then define the first plane for triene system using three atoms of C8, C9 and C10; while the second plane for benzene group of Y378 using three atoms of CE1, CE2 and CG. Surprisingly, not a parallel but a displaced T-shape interaction between two conjugate systems was found, based on the calculated dihedral angle of 68.6 ± 20.9°. This kind of topology for two π conjugate systems is not unique. Indeed, a displaced T-shaped topology for benzene dimer system has been suggested to be stable by Hobza and coworkers.58, 59, 60 If we carefully examine the geometry of Y378 and triene conjugate system, we might simply postulate that the Y378 could hinder the rotation at C7 site via this special displaced T-shaped π-π interaction and thus maintain its initial enantiomer feature during the reaction. However, a recent study26 on LTA4H from the African claw toad, Xenopulaevis (xlLTA4H) indicates that a phenylanaline residue (F375) occurs at the equivalent position. Two products, LTB4 and its isomer of ∆6-trans-∆8-cis-LTB4 (show in Figure 6), can be identified during the reaction although LTB4 is the main product. More interestingly, the F375Y could abolish the formation of the isomer, ∆6-trans-∆8-cis-LTB4. It was then assumed that interaction between Y378 and Y383 might be another key factor that could affect the enatioselectivity of product.61 Indeed, we can really locate this hydrogen bond evidenced by

!(%&)!(%)

= 2.89 ± 0.20 Å in our simulation. Therefore, our 12


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simulation is in good agreement with experimental proposal. In order to understand the enantioselectivity functional role of Y378 in the formation of final product of LTA4H, we further carried out two more site directed mutagenesis simulations of Y378F and Y378Q. MD setup protocols are essentially the same as we did for the wild type (WT) LTA4H, but the simulation time is 2ns for each mutated system. First of all, the substrate can stay stably at the active site with the same tetra-coordination status for Zinc ion. The torsion angels of the triene system for Y378Q and Y378F are summarized in Table 3. Clearly, the torsion angles show little variation upon mutations at the 378th position. Although such short MD simulations might not be long enough to cause large conformational change for the triene system, relatively stable conformation might also suggest low yield of the second enantiomer like ∆6-trans-∆8-cis-LTB4 when a phenylalanine residue occurs at this equivalent position for xlLTA4H. In addition, we further calculated the dihedral angle between C7-C12 triene system and the benzene group of F378, which is about 98.45° ± 32.97°. Much larger fluctuation than that in WT case indicates the triene system might have more chance to undergo some conformational changes. Combining with experimental observances, we could still suggest deviation from the displaced T-shaped π-π interaction around triene system might cause loss of its enantioselectivity for LTA4H. Of course, extensive electronic structure calculation and reactive mechanism simulation are required for detailed understanding the functional role of Y378. Current MD simulation could only provide some geometrical hints for subsequent investigations. Further extensive reactive mechanism simulation is highly desired to address this interesting issue. Another important issue related to the generation of LTB4 is the nucleophilic attacking (NA) reaction at C12 atom. During this process, it has been suggested that D375 could activate one nearby water molecule by abstracting its hydrogen atom to generate hydroxyl group, which will further attack C12 atom to fulfill the formation of LTB4. This NA reaction could also ameliorate the positive charge developed on C6 atom after the initial ring-opening reaction. Therefore, binding patterns around C12 atom deserve further investigation. Throughout the MD simulation, the D375 is 13



stabilized by H139 via a stable hydrogen bond, evidenced by '('&)⋯ ( ) of 2.15 ± 0.60 Å. On the other hand, one nearby water molecule could be located during the MD simulation, which is further stabilized by D375 via the hydrogen bond with ' ('&)⋯ ( ) = 2.87 ± 0.44 Å. At the same time, this water molecule stays 3.86±0.58 Å away from the C12 site. Such structural patterns could further guarantee the possibility of a subsequent NA reaction. 3.3 Ring Opening Reaction in Epoxide Hydrolase Mechanism We have constructed Michaelis complexes of LTA4H for both peptidase and epoxide hydrolase mechanisms. As we have mentioned above, the peptidase mechanism will not be further discussed here since it has been investigated to some extent. We will then focus on the epoxide hydrolase mechanism, however, only the first step of the epoxy ring opening reaction will be investigated in this work. The reason is to check the Michaelis complex we built and provide more insights into the complete catalytic reaction. Since the active site water molecule within epoxide hydrolase mechanism shows a little bit far away from epoxy ring, the first step of ring opening should be activated solely by zinc ion. However, we still confirmed this by a two-dimensional potential energy surface calculation including both dissociation of C6-OB bond and proton transfer involving E271. According to Figure S2, two reaction directions are generally independent. Thus, the reaction coordinate is then defined as the bond distance of C6-OB with a range of [1.52, 2.32] Å. First of all, we calculated the minimum energy path (MEP) using the so-called reaction coordinate driving method. The MEP is optimized by several times with a forward to backward strategy. To include entropic contribution, we further calculated the potential of mean force (PMF) for this reaction. Calculated PMF curve is plotted in Figure 7 with the barrier height of 8.5 kcal/mol. The snapshots for all three stationary states are plotted in Figure 8, and selected geometric parameters are given in Table 4. The first step of the whole hydrolysis reaction is quite simple, which is the bond dissociation of C-O bond. The (# = 1.92 Å is found for the transition state and 14


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2.12 Å for the intermediate. During the cleavage of this C-O bond, zinc ion acts as a Lewis acid catalyst, evidenced by the distance between OB and Zn shortened from 2.22 to 2.16 Å. The newly developed positive charge on C6 atom can be further stabilized and delocalized via the π-conjugate triene system (C7-C12). Indeed, the bond distance of C6-C7 is shortened by about 0.07 Å. Such kind of charge delocalization could increase the charge on C12 atom, which should facilitate the subsequent NA reaction by nearby water molecule. Once the dissociation of C-O bond completes, the active site water molecule, which is hydrogen bonded with the protonated E271, moves inside and becomes close to the epoxy atom (OB). It should be pointed out here that the optimized reactant structure is somewhat different from the structure obtained by our MD simulation, esp., in the position of the active site water molecule. This is because our MEP calculations were carried out several times using the forward-and-backward strategy along with reaction coordinate using adiabatic mapping approach. It could thus be understandable that the position of this water molecule changes so large. We should emphasize here such a conformer is also ready for the subsequent reaction of hydrogen migration from the water to the substrate, in which E271 will serve as the general acid catalyst to activate this water. Basically, the generation of LTB4 after the first step of epoxy ring opening needs two other key processes. One is above mentioned protonation transfer (PT) from the water to the epoxy oxygen atom, and the second one is the NA reaction at C12 atom by the nearby water. To understand the possible reactive site after the ring opening reaction, the frontier orbital analyses were applied for the intermediate. For simplicity, we then carried out a truncated model calculation including QM region atoms at B3LYP/6-31+G(d,p) level of theory. Corresponding HOMO and LUMO for the truncated model are displayed in Figure 9. As we can see, both HOMO and LUMO mainly distribute on the triene system and D375, but some distinct differences can be seen. There are some electron distributions on the water molecule (close to D375) for HOMO, whereas no such population can be found for LUMO. More importantly, we cannot find obvious electron distribution on the side chain carboxylate group of E271 no matter HOMO or LUMO, although the epoxy oxygen atom has some electron 15



distributions. All of these can suggest that the subsequent step might prefer NA reaction first. This NA reaction could occur at C12 atom attacked by the water molecule via proton abstraction of D375. This is reasonable since the developed carbenium ion at C6 atom after ring-opening is metastable. The NA reaction could largely neutralize the positive charge and lower the total energy. Of course, further extensive reaction mechanism simulations are required to elucidate this mechanistic issue, esp., from the quantitative angle. The proposed epoxidase mechanism is finally summarized in Scheme 3. Finally, as shown in Figure 7, the obtained intermediate seems to be a meta-stable status, since the barrier height back to reactant is about 0.5 kcal/mol. Clearly, the newly developed carbenium ion at C6 is in a metastable status, although the charge can be stabilized and delocalized through the whole C7-C12 triene system. Of course, the subsequent reactions of PT and NA could largely release this energy. 4. Conclusions In this work, for a bifunctional enzyme, LTA4H, SCC-DFTB/CHARMM method was employed to address different binding patterns for both peptidase and epoxide hydrolase mechanisms. Two mechanisms share one common active site. There are three same ligands to zinc ion provided from enzyme, but the fourth ligand is different between two mechanisms. More importantly, the L-shaped active site is very suitable for the binding of the long fatty acid tail of the substrate LTA4, while two positively charged residues, R563 and K565, are shown to be useful in stabilization of the substrate and even restricting the length of polypeptide substrate. When it functions as a peptidase, the substrate (R1'-S2'-R3') binding mode is essentially the same as other aminopeptidases. The fourth ligand to Zinc ion is one water molecule, which is also hydrogen bonded with a downstream E296. This water molecule is close to the scissile C-N bond and ready for the NA reaction. Therefore a common adapted GAGB mechanism was proposed in Scheme 1 for LTA4H as a peptidase. In particular, E296 has the double functions of general base and general acid. When it functions as an epoxide hydrolase, we then docked its natural substrate 16


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of LTA4 into the active site. During the MD simulation, the fourth ligand to Zinc ion is the epoxy oxygen atom. On the other hand, we have confirmed that E271 should be protonated, which is further hydrogen bonded with a water molecule. The C7-C12 triene system remains coplanar topology. At the same time, the phenyl hydroxyl group of Y378 is found to form a displaced T-shaped π-πinteraction with this triene system, which might be the reason to determine the enatioselectivity of final product. In this work, we only carried out simulation for the first step of epoxy ring opening reaction, which results a metastable intermediate. The frontier orbital analyses show that the NA reaction at C12 atom rather than PT reaction at epoxy oxygen atom should occur first. Subsequent reaction mechanism simulations are underway in our lab to get a complete understanding of epoxide hydrolase mechanism for LTA4H.

Acknowledgement This work was funded by the National Key R&D Program (No. 2016YFB0700801) and the National Natural Science Foundation of China (No. 21473117). Some of the results described in this work were obtained on the Supercomputing Center of Chinese Academy of Science.

Supporting Information Distance between OB and the oxygen atom of the active site water. Two-dimensional potential energy surface for the first step of epoxy ring opening reaction. Full citations of Ref. 13 and 43. This material is available free of charge via the internet at http://pubs.acs.org/.

Reference 1.

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56. Andberg, M.; Wetterholm, A.; Medina, J. F.; Haeggstrom, J. Z., Leukotrieen A4 Hydrolase: A Critical Role of Glutamic Acid-296 for the Binding of Bestatin. Biochem. J. 2000, 345, 621-625. 57. Wetterholm, A.; Medina, J. F.; Radmark, O.; Shapiro, R.; Haeggstrom, J. Z.; Vallee, B. L.; Samuelsson, B., Leukotriene A4 Hydrolase: Abrogation of the Peptidase Activity by Mutation of Glutamic Acid-296. Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 9141-9145. 58. Hobza, P.; Selzle, H. L.; Schlag, E. W., Potential Energy Surface of the Benzene Dimer: Ab Initio Theoretical Study. J. Am. Chem. Soc. 1994, 116, 3500-3506. 59. Hobza, P.; Selzle, H. L.; Schlag, E. W., Potential Energy Surface for the Benzene Dimer. Results of Ab Initio CCSD(T) Calculations Show Two Nearly Isoenergetic Structures: T-Shaped and Parallel-Displaced. J. Phys. Chem. 1996, 100, 18790-18794. 60. Spirko, V.; Engkvist, O.; Soldan, P.; Selzle, H. L.; Schlag, E. W.; Hobza, P., Structrue and Vibrational Dynamics of the Benzene Dimer. J. Chem. Phys. 1999, 111, 572-582. 61. Andberg, M. B.; Hamberg, M.; Haeggstrom, J. Z., Mutation of Tyrosine 383 in Leukotriene A(4) Hydrolase

Allows

Conversion

of

Leukotriene

A(4)

into

5s,

6s-Dihydroxy-7,

9-Trans-11,

14-Cis-Eicosatetraenoic Acid - Implications for the Epoxide Hydrolase Mechanism. J. Biol. Chem. 1997, 272, 23057-23063.

21



Scheme 1. The proposed peptidase mechanism of LTA4H.

22


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Scheme 2. Two putative mechanisms of LTA4H as an epoxide hydrolase. (A) One active site water molecule is suggested to be the fourth ligand to Zinc ion.3, 4, 11. (B) The oxygen atom on epoxy ring B is supposed to be the fourth ligand to Zinc ion.10, 25, 29 .

23



Scheme 3. Proposed mechanism of LTA4H with epoxide hydrolase activity.

24


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Figure 1.The binding environment of LTA4.

25



Figure 2. Putative interactions between enzyme and substrates and corresponding atomic definitions. Left panel denotes the enzyme-peptide complex, and the right panel represents enzyme-LTA4 complex.

26


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Figure 3. RMSDs of protein backbone atoms for (A) LTA4H/R1′-S2′-R3′ complexand (B) LTA4H/LTA4 complex as a function of time.

27



Figure 4. (A)Snapshot of LTA4H/R1'-S2'-R3' complex extracted from the MD trajectory, (B) Overlap representation of active sites among LTA4H (carbon in green), ACE (carbon in cyan) and TLN(carbon in yellow). (C) Overlap representation of active sites between LTA4H (carbon in gree) and CPA (carbon in blue).

28


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Figure 5. Snapshot of LTA4H extracted from MD trajectory as an epoxide hydrolase. Active-site are colored with purple, ligand is showed in yellow. The blue color represents the Y383 and Y378 residue. An explicit hydrogen bond between Hε2 and water is showed.

29



Figure 6. LTB4 and its isomer structure.

Figure 7. PMF of the ring-open reaction via the epoxide hydrolase activity. 30


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Figure 8. The stationary states of ring-opening reaction via the epoxide hydrolase activity.

31



Figure 9. The frontier molecular orbital analyses for the enzyme-intermediate complex after ring-opening reaction. Zinc atom is shown in purple color, nitrogen atoms are in blue, and oxygen atoms are in red.

32


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Table 1. Selected key geometries for recognition of R1'-S2'-R3' by LTA4H. For comparison, corresponding values from X-ray structure (PDB id 3B7S) are also included. Distance (Å) Zn···OH2(wat) Zn···Nε2(H299) Zn···Nε2(H295) Zn···Oε1(E318) H2(wat)-Oε1(E296) HN1(R1') ···Oε1(E271) HN2(R1') ···Oε1(Q136) HN3(R1') ···Oε2(E318) Hε(R1') ···Oδ1(D375) O(R1') ···Hη(Y383) Hγ1(S2') ···Oε1(E296) HN(S2') ···O(G269) O(S2') ···HN(G268) OT1(R3') ···Hη22(R563) OT2(R3') ···Hε(R563) a

MD 2.07±0.06 2.02±0.06 2.00±0.06 2.06±0.07 2.25±0.36 3.43±1.14 2.70±0.74 2.46±0.60 3.40±0.24 1.69±0.10 2.56±1.10 1.93±0.18 1.98±0.39 1.69±0.10 1.75±0.11

distances between N-O; distances between O-O.

b

33


Exp.12 2.03 2.08 2.00 3.12a 4.24a 3.08a 6.26a 2.62b 2.86b 2.91a 2.78a 2.75a 2.97a


Table 2. Selected key geometries for recognition of LTA4 by LTA4H Name Zn···OB Zn···Nε2(His 295) Zn···Nε2(His 299) Zn···Oε1(Glu 318) OB···Hη(Tyr 383) O2···Hη22(Arg 563) O1···Hε(Arg 563) O1···HN(Gly 268) Hη(Y378) ···Oη(Y383) OH2(wat)···OB

QM/MM MD 2.27±0.24 1.98±0.05 1.98±0.05 2.22±0.40 2.44±0.48 1.85±0.31 1.92±0.40 2.01±0.40 2.89±0.20 6.47±2.63

34


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Table 3. Statistically averaged dihedral angles of the triene system for WT, Y378F and Y378Q through MD simulations*. Dihedral angles WT Y378F Y378Q (degree) -11.5±22.6 -0.5±11.4 -0.5±11.4 -. 174.4±4.3 174.1±3.5 171.1±3.6 - -170.8±7.0 -170.9±5.4 -171.1±5.4 - -173.9±3.7 -173.9±3.7 -174.2±4.3 - -/ 170.6±7.7 170.3±5.7 170.7±5.5 1.4±9.0 -0.3±7.34 -2.2±7.6 - *Definitions of all dihedral angles: -. : OB-C6-C7-C8; - : C6-C7-C8-C9; - : C7-C8-C9-C10; - : C8-C9-C10-C11; -/ : C9-C10-C11-C12; - : C10-C11-C12-C13.

35



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Table 4.Selected geometric parameters for the ring-opening reaction via epoxide hydrolase mechanism catalyzed by LTA4H. Distances (Å) ES TS EI Angles (degree) C6-OB

1.52

1.92

2.12

C5-C6

1.48

1.48

1.49

C5-OB

1.51

1.49

1.48

C6-C7

1.47

1.42

1.40

OB-Zn

2.22

2.16

2.12

Zn-Nε2(H295)

1.98

1.99

1.99

Zn-Nε2(H299)

1.97

1.98

1.98

Zn-Oε1(E318)

2.01

2.02

2.03

OH2-Hε2(E271)

1.85

1.87

1.87

H1-Oε2(E318)

1.78

1.78

1.78

H2-OB

2.33

2.23

2.09

H8-C6-C7-H9

-30.48

-24.14

-17.40

φ0

14.69

27.47

30.67

φ1

-176.98

179.43

176.64

φ2

170.16

166.78

167.68

φ3

-177.23

179.95

179.69

φ4

174.25

172.20

170.58

φ5

0.36

-1.12

-1.99

36


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TOC Graphic

37


MM Molecular Dynamics Investigations of the Substrate Binding

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