Catalytic Mechanism of Hyaluronate Lyase from Spectrococcus

Article pubs.acs.org/JPCB

Catalytic Mechanism of Hyaluronate Lyase from Spectrococcus pneumonia: Quantum Mechanical/Molecular Mechanical and Density Functional Theory Studies Min Zheng and Dingguo Xu* MOE Key Laboratory of Green Chemistry and Technology, College of Chemistry, Sichuan University, Chengdu, Sichuan, 610064, P. R. China S Supporting Information *

ABSTRACT: Hyaluronate lyase from Spectrococcus pneumonia can degrade hyaluronic acid, which is one of the major components in the extracellular matrix. The major functions of hyaluronan are to regulate water balance and osmotic pressure and act as an ion-exchange resin. It has been suggested in our previous molecular dynamics simulation that the binding of the substrate molecule could lead to the ionization of Y408 and protonation of H399. Followed by our recent molecular dynamics simulation of the enzyme− substrate complex, a unified proton abstraction and donation mechanism for this enzyme can be established using a combined quantum mechanical and molecular mechanical approach and density functional theory method. Y408 is shown to serve as the general base in the proton abstraction, while general acid is the next proton donation step. Overall, this reaction can be classified into synelimination reaction mechanism. The neutralization effects of C5 carboxylate group by several polar residues such as N349 and H399 were also examined. Finally, in combination of our previous molecular dynamics simulations, a complete catalytic cycle for the degradation of hyaluronan tetrasaccharide catalyzed by the hyaluronate lyase from Spectrococcus pneumonia is proposed. Scheme 1. General Mechanism (syn and anti-Elimination) for the Degradation of Uronic Containing Polysaccharides Catalyzed by PLsa

1. INTRODUCTION The degradation of glycosidic linkage is one of the most important reactions in the natural world. Two types of enzymes, glycoside hydrolases (GHs)1−3 and polysaccharide lyase (PLs),4,5 are responsible for such catalytic processes but with different mechanisms. With assistance of water molecule in the active site, the GHs can depolymerize the polysaccharides via either retention or inversion mechanisms according to the configuration change at the C-1 anomeric carbon center.3,6,7 On the other hand, PLs can degrade the uronic acid-containing polysaccharides via a β-elimination mechanism to generate an unsaturated hexenuronic acid residue.5 On the basis of amino acid sequences, the PLs currently are classified into 22 subfamilies. Detailed information including sequences or crystal structures entry codes can be found in the Web site of the CAZy (Carbohydrate-Active Enzymes) database (http://www. cazy.org).8 For the nonhydrolytic mechanism to degrade the glycosidic bond catalyzed by lyases, it has been discussed a lot. Generally, a unified multistep reaction processes, namely, protonabstraction and donation (PAD), has been summarized by Gacesa in 1987.9 The reaction is involved with the neutralization of the substrate C5 carboxyl functionality, proton abstraction at C5, and syn or anti elimination of the 4-Oglycosidic bond to form a C4−C5 double bond. The general mechanisms are summarized in Scheme 1. Clearly, it is basically a general base/general acid mechanism. On the other hand, the © 2013 American Chemical Society

a

The blue ball represents the neutralization groups, either metal ions or positively charged residues.

Received: June 23, 2013 Revised: August 6, 2013 Published: August 14, 2013 10161

dx.doi.org/10.1021/jp406206s | J. Phys. Chem. B 2013, 117, 10161−10172

The Journal of Physical Chemistry B

Article

Scheme 2. Atomic Definitions and Possible Interactions between Enzyme and the Tetrasaccharidea

a

The QM region is labeled using red color. Dash lines represent possible hydrogen bond connections.

of PL8, complexed with a substrate molecule, in which they suggested that the active site tyrosine residue (Y255) should serve as the general base and general acid throughout the reaction. Based on a recent X-ray structure for Streptomyces coelicolor A3 HL, Elmabrouk et al. suggest that the tyrosine (Y253) should serve as the general base to abstract the proton from the C5 of the GlcUA residue since this tyrosine lies in close proximity to the +1 subsite.28 Our recent QM/MM MD simulations11 have demonstrated that there is practically no energy barrier to form the enzyme−substrate complex (ES) for the hyaluronan degradation catalyzed by the hyaluronan lyase from S. pneumonia. The active site tyrosine (Y408) is proposed to be in its ionized state with the binding of substrate molecule. Meanwhile, the nearby histidine residue (H399) is then protonated to neutralize the C5-carboxyl group. Such special substrate binding characteristics strongly support the later mechanism that the tyrosine residue acting as the general base/ general acid as shown in Scheme 3 in ref 11. There are few theoretical investigations on the mechanism for polysaccharide lyases reported so far. Detailed simulations from the microscopic angle might provide some insightful understanding for those enzyme systems. We report here a detailed theoretical investigation on the catalytic mechanism of the SpnHL in the degradation of hyaluronan tetrasaccharide molecule. We will in this work try to address several key issues related with the mechanism, such as the detailed reactive processes, whether or not the neutralization of C5-carboxylate group is really that important. Another interesting issues for understanding a reaction mechanism involving catalysts is how the catalysts restore their original form after a complete reaction. Combined our previous simulation, we will in this work propose a complete pathway for the change of enzyme catalyst.

neutralization of the C5-caboxylate group is important to facilitate the catalysis. In this work, we will focus on the hyaluronan lyase from Spectrococcus pneumonia (SpnHL).10 This is a continuous work following the Michaelis complex model constructed in our recent combined quantum mechanical and molecular mechanical (QM/MM) simulations.11 SpnHL belongs to PL subfamily 8, which has an overall α/α + β architecture. The SpnHL can degrade hyaluronic acid (HA),12−14 which is one of the major components of the extracellular matrix.15 The HA is an anionic, nonsulfated glycosaminoglycan, which is composed of repeating disaccharide unit linked together via a β-1,3-glycosidic bond. Each disaccharide unit consists of D-glucuronic acid (GlcUA) and Nacetyl-D-glucosomine (GlcNAc). HA is widely expressed throughout connective epithelial and neural tissues. The major functions of hyaluronan are to regulate water balance and osmotic pressure and act as an ion-exchange resin. Hyaluronan has been shown its wide applications in the skin wound repair processes.16 It is often used as a tumor monitor for cancer development.17 Indeed, it has been shown that the degradation mechanism of hyaluronan lyases is critical in many physiological processes, and the inhibitors studies to bacterial hyaluronidase are important to pharmaceutical development.18−21 Besides SpnHL, there are three other members in this subfamily, such as chondroitin AC lyase (EC 4.2.2.5),22 xanthan lyase (EC 4.2.2.12),23 and chondroitin ABC lyase (EC 4.2.2.20).24 These structures obviously can provide sufficient information to understand the substrate binding pattern and plausible catalytic mechanisms for members in PL8 subfamily. One of the major controversies around understanding of the catalytic mechanism is the candidate of the general base, and thus different catalytic mechanisms were proposed for members in the PL8 subfamily. First of all, based on the fact that histidine residue is a better general base than tyrosine residue and the Xray structure of the enzyme, Jedrzejas and co-workers10,25−27 proposed that H399 acts as the general base, N349 has the function of neutralizing C5-carboxylate group, and Y408 is the proton donor to transfer proton to glycosidic bond. Later, Lunin et al.22 argued this proposal according to some kinetic data analyses; for example, H399A still has some significant activity, while mutation of Y408F completely deactivates the enzyme. Maruyama et al.23 also independently reported a structure for Bacillus sp. GL1 Xanthan lyase, another member

2. COMPUTATIONAL DETAILS 2.1. QM/MM Models. It has been widely accepted that the QM/MM approach29 is one of the suitable tools to simulate the system-like enzymes. Based on the scenario of divide-andconquer, the total system is divided into two parts. The smaller region containing those atoms involved in the reaction will be treated using high level quantum mechanical method, thus has the name of QM region. On the contrary, the rest of the atoms (MM region) including the surrounding environment protein and solvent atoms will be described using a force field. In this 10162



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substrate. To confirm the proposed mechanism, we first extracted some snapshots from the MD trajectory of the enzyme−substrate (ES) complex. Since the first step of elimination involves several bond forming and bond breaking processes, we then calculate a two-dimensional potential energy surface using the so-called adiabatic mapping approach (sometimes referred to reaction coordinate driving method41) to account for most of the reactive information. Corresponding reaction coordinates are then defined as d1 = dC5−H5 − dOH(Y408)−H5 and d2 = dC4−O4, which are related with the abstraction of hydrogen H5 and the cleavage of glycosidic bond C4−O4, respectively. The second step is a simple proton transfer from Y408 to O4 atom, and the reaction coordinate can be given as d3 = dOH(Y408)−H5. The obtained minimum energy paths (MEPs) are further refined using the conjugate peak refinement (CPR) approach.42 It has been suggested that CPR corrected energy activation barrier height can be sufficiently close to experimental value, especially the proton transfer reactions.43 The reaction coordinate in CPR is then defined as

work, the CHARMM2230 all-atom force field is applied to express the MM region. As discussed in many work, the selection of the quantum mechanical method in the electrostatic structure calculations of QM is critical for the reliability of the simulation. In this work, the self-consistent charge density tight binding method (SCCDFTB),31,32 which has been implemented to CHARMM by Cui et al.,33 was applied to all of QM/MM simulations. The combined SCC-DFTB/MM approach has been shown its success in some important enzyme systems including glycoside hydrolases.34,35 The initial model was adopted using the ionized model in our recent simulation of the substrate binding model of hyaluronate lyase from S. pneumonia complexed with a tetrasaccharide molecule.11 As shown in our previous paper, such a model is obtained by a spontaneous proton migration from Y408 to H399 with the substrate binding. Detailed setup protocol for the molecular dynamics simulation has been described in the previous work. Here we just give a simple description. The atomic definitions and possible interactions between enzyme and the hyalurona tetrasaccharide molecule are given in Scheme 2. HA1 and HA2 are defined as the consecutive positions of hyaluronan disaccharide within the tetrasaccharide substrate numbered from the reducing toward the nonreducing end. The nomenclature of each atom for enzyme is adopted from CHARMM definition. Particularly, in accordance with our previous simulation, the proton on H399 (migrating from Y408) will be still taken using the name of HH. The overall system is solvated in a pre-equilibrated TIP3P36 water sphere with 25 Å of radius centered at C5 (HA1) atom. A stochastic boundary condition37 is applied to reduce the computational costs. During the simulation, if not otherwise stated, the SHAKE algorithm38 is applied to keep all covalent bond involving hydrogen atoms except H5 atom of HA1. The link atom approach39 was applied to describe the covalent interface between QM and MM regions. In all molecular dynamics simulation, the integration step is set as 1 fs. A group-based switching scheme was applied for nonbonded interactions.40 Since the ionization status of Y408 and H399 has been determined in aforementioned QM/MM simulations, we will then take that model as the starting point in our current reaction mechanism simulations. Following our recent work, the QM region consists of the side chains of Y408, N349, R462, H399, and the entire substrate of hyaluronan tetrasaccharide. The total number of atoms in QM region is 155, and the total charge of the QM region is −1. The inclusion of R462 is important, since it provide a salt bridge to stabilize the putative general base/general acid of ionized Y408. For the Michaelis complex obtained by us,11 we can find that a stable hydrogen bond between the H5 atom of HA1 and the OH atom of Y408 is formed during the QM/MM molecular dynamics evidenced by the distance of 1.94 ± 0.12 Å. Based on this structural characteristic, we further proposed a unified syn-elimination mechanism that will be shared for members in the polysaccharide lyase subfamily 8. The first elimination step involves a proton (H5) transferring to OH atom (Y408), cleavage of glycosidic bond, and formation of a unsaturated disaccharide (generation of C4−C5 double bond). The subsequent proton transferring from Y408 to O4 atom will finally restore the ionized status for enzyme before reaction. Y408, in this case, is postulated to be the general base/acid catalyst in the degradation of the hyaluronan tetrasaccharide

i=2

λ=

∑ {|x (⃗ i) − x (⃗ i − 1)|}/ N

3n (1)

where N is the order of the points along the CPR trajectory, x(i) is the coordinate of point i, and n is the number of atoms. Similar with our previous work,44 λ was normalized throughout this work if not otherwise stated, so that 0 ≤ λ ≤ 1. However, minimum energy path profiles are not sufficient to describe the activation energy barrier height for the reaction occurring in the enzyme systems, since such methods do not include entropic contributions provided by protein environment. To tackle this problem, we then computed the potentials of mean force (PMFs) to include the fluctuation of the system. The structures obtained in the MEP calculations along the reaction coordinates were used as the initial structures in the PMF calculations. In the PMF calculations, umbrella sampling45 was applied to enhance sampling around the peak region for each sampling windows. For the first elimination step, there are 563 windows with harmonic constraints around 100−300 kcal/(mol·Å2). For the following protonation step, there are 18 windows, and a harmonic constraint of 50 kcal/ (mol·Å2) for each window was applied. A total of 100 ps constrained MD simulations were performed for each window with the first 60 ps heating to 300 K and equilibrium, and the rest of the 40 ps trajectory was saved for data analysis. Finally, the weighted histogram analysis method (WHAM)46,47 was used to obtain the PMFs for two steps. 2.2. Truncated Active-Site Models. To provide an independent check on the QM/MM models described above, we further performed a high-level density functional theory (DFT) study of a truncated active-site model. The truncated model consists of a shortened substrate and the analogue of side chain groups of three residues (His, Asn, Tyr). In such a model, the His, Asn, and Tyr residues were approximated by methyl imidazole, acetamide, and 4-methyl-phenol, respectively. To reduce computational cost, here we use a trisaccharide units (GlcNAc-GlcUA-GlcNAc) to mimic the tetrasaccharide substrate. The total number of atoms for the DFT calculation resulted in 103. The initial guess of the reactant complex (RC) is adopted using the structure obtained by our QM/MM simulation. 10163



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Figure 1. Computational models for proton affinities used in this work: (a) GlcUA, (b) ASN-GlcUA, (c) ASN-GlcUA-HSP, (d) Ca2+-GlcUA, (e) Ac-Tyr-OCH3, (f) GlcNAc. For clarification, we include models of both B and BH+ in the figure.

trends for those simulated models are still consistent between the semiempirical and high-level methods. The gas phase proton affinity (PA) of a compound B is defined as the negative standard reaction enthalpy of protonation at 298.15 K. It can be calculated using the following scheme:53

All stationary states are fully optimized using the Becke3− Lee−Yang−Parr (B3LYP) exchange-correlation functional48,49 with a standard basis set (6-31G(d,p)). Harmonic frequency calculations were performed to confirm the minima (all positive) and transition states (only one imaginary frequency). Intrinsic reaction coordinate (IRC)50 calculations were carried to connect all of stationary states. To account for the solvent effects, the polarized continuum model (PCM)50 was applied both in the protein environment (ε = 5) and in water (ε = 80). All of DFT calculations are performed using Gaussian09 suite of program.51 2.3. Proton Affinity Calculations. Since the degradation of hyaluronan tetrasaccharide molecule is dominated by several proton transferring processes,10,11,25 it would be necessary to examine corresponding proton affinities (PAs) for several important molecule species involving in the reaction. We then constructed six models as depicted in Figure 1, for which the PAs were calculated at both SCC-DFTB and B3LYP/6-311G+ +(3df, 2p) levels of theory, respectively. Many previous computational studies have indicated that semiempirical methods are not ideal to predict accurate PAs or pKa values for those titratable groups.52,53 B3LYP/6-311++g(3df, 2p) has been proved to be one of the methods to give a reasonable proton affinity for model systems.54 However, it still deserves to investigate the performance of SCC-DFTB in the calculation of PAs. As shown in our recent work, the computational errors of SCC-DFTB for the PAs are constant 14 kcal/mol systematic difference from both DFT and experimental data.53 The overall

B + H+ → BH+

(2)

PA = −[ESCF(BH+) − ESCF(B) + Evib(BH+) − Evib(B)] 5 + RT (3) 2

in which ESCF represents the electronic energy obtained from SCF calculations; Evib includes the zero-point energy and temperature corrections to the vibrational enthalpy; 5/2 RT denotes the contributions from translational energy of proton and the ΔPV term. For SCC-DFTB, the PA can be calculated as: PA = −[ESCF − DFTB(BH+) − ESCF − DFTB(B) + Erep[ρ0H ] + Evib(BH+) − Evib(B)] +

5 RT 2

(4)

In this work, we calculated the proton affinities using the value of −141.9 kcal/mol53 for Erep[ρH0 ]. The vibration term Evib will not be considered here due to the comparative feature of the calculations. 10164



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proton affinity of C5 atom about 104.1 kcal/mol (SCC-DFTB) and 95.6 kcal/mol (DFT), respectively. This leads to corresponding PASCC‑DFTB = 365.5 kcal/mol and PADFT = 358.0 kcal/mol, respectively. They can be roughly comparable with PA values of the tyrosine model as shown in Table 1. Obviously, such results could partially suggest that the existence of a positive group to neutralize the C5 carboxylate group can significantly increase the reactivity of C5−H5 bond. With the inclusion of neutralization groups, the PA gap between C5 atom and model tyrosine OH atom is dramatically reduced to several kcal/mol at SCC-DFTB level, while even closer (less than 1.0 kcal/mol) at the DFT level of theory if considering the vibration contributions. Since we just calculated gas-phase PA values and no protein environment is included, some differences from the real case might be expected. However, from current calculations, we can easily see that the inclusion of suitable neutralization group does show the function of polarizing H5 atom. This is of course critical to facilitate the subsequent degradation of glycosidic bond. The PA calculations can be used to answer why the lyases need the so-called neutralization group. From other words, the proton transfer from Y408 to H399 along with the substrate binding suggested in our previous simulation seems to be one of the key steps for the whole catalytic reaction. Such process perfectly satisfies several mechanistic prerequisites for the subsequent reaction: a reasonable enzyme−substrate complex ready for subsequent reaction, a putative general base/general acid (Y408), and a positive H399. Once again, our simulation seems to be against the mechanistic suggestions by Jedrzejas and co-workers,10,25,26,55 since the requirement of a positive H399 clearly cannot be the general base in the subsequent reaction. More detailed reaction simulations are required to get complete understanding of the energetic profiles for the catalytic reaction.

Computational models used for the proton affinity calculations are based on the mechanism proposed by us, in which the overall reaction are dominated by two steps of proton transfer, that is, H5 transferring to Y408 and finally to glycosidic oxygen atom of O4. It is well-known that the σ bond of C−H in general is unreactive. This could clearly bring up one critical issue which is the reason of proton (H5) transferring from C5 to OH atom of Y408 or H399 as suggested by researchers. It has been suggested that one major function of the PLs is to provide neutralization groups (either metal ions or polar residues) at the substrate C5 carboxylate group. Further, such function is postulated to acidify the H5 atom and then facilitate the reaction. Can we quantify this kind of contribution? Therefore, several models were constructed to investigate the effects of various neutralization groups on PA of C5 atom, especially side chain groups of Asn and Hsp. The reason that we investigate the positive histidine residue is the H399 is assigned to positive state to neutralize the carboxylate group in our recent work. It would be interesting to investigate the PA for the side chain of the ionized tyrosine residue and glycosidic oxygen atom, since they are essential in the second step reaction. All of six computational models shown in Figure 1 were then built to address this issue. Calculated PAs for model systems are listed in Table 1 obtained at both SCC-DFTB and DFT levels of theory. For Table 1. PA Values (kcal/mol) Calculated with DFT (B3LYP/6-311++G(3df, 2p)) and SCC-DFTB Methodsa model

SCC-DFTB

DFT (without Evib)

DFT (with Evib)

NBO charge (C5)

GlcUA ASN-GlcUA ASN-GlcUAHSPb Ca2+-GlcUA Ac-Tyr-OCH3 GlcNAc

489.4 469.6 365.5

470.9 453.6 358.0

460.0 443.6 345.2

−0.136 −0.134 −0.085

272.6 352.9 349.2

265.5 344.3 340.6

0.022

359.5 364.3

3. RESULTS AND DISCUSSION 3.1. Reaction Path. In our early work,11 the QM/MM MD simulations have suggested that the active site tyrosine (Y408) is in its ionized state with the binding of the substrate molecule. At the same time, the phenylhydroxyl hydrogen atom (HH) of Y408 will transfer to the nearby histidine residue (H399) via a nearly barrierless transition state. The positively charged H399 is then hydrogen bonded with the C5-carboxyl group to serve as the so-called neutralization group as suggested for lyases. Such geometrical characteristics then lead to generate the ES complex for subsequent elimination reaction. Since we have given a detailed description for the initial reactive conformer in our recent work,11 we will, in this work, just use it as the initial structure for the investigations of subsequent reaction simulation. As suggested by Gacesa,9 the overall degradation of polysaccharide catalyzed by lyases might proceed via the socalled PAD mechanism. On the basis of our QM/MM MD simulation,11 we have identified that the ionized Y408 is the only candidate in the active site to serve as the general base, and experimental evidence also indicates that the site-directed mutagenesis study for Y408 could completely deactivate the activity. Therefore, we first calculated a two-dimensional (2-D) potential energy surface (PES) with respect to the proton abstraction (H5 to Y408) and cleavage of glycosidic bond (C4−O4). The calculated 2-D PES is plotted in Figure 2A, with the energy barrier height calculated to be 21.8 kcal/mol. Only one transition state (TS1) can be located, and the intermediate

a

NBO charges at B3LYP/6-311++G(3df, 2p) level of theory for C5 atom are included. Ca2+-GlcUA represents a model to calculate the PA for C5 atom using the Ca2+ as the neutralization group to C5 carboxylate group, which is shown in Figure 1d. Ac = acetyl-protecting group. bHSP: protonated histidine residue.

more detail, we further calculated the NBO charge for C5 atom to see its change along the addition of neutralization group. As shown in Table 1, as large as 10 kcal/mol differences between SCC-DFTB and DFT methods can be observed, which is similar with our recent work on angiontensin converting enzyme (ACE)44 and previous suggestions.53 It is remarkably noted that the inclusion of neutralization group at C5carboxylate group does significantly lower the PA of C5 atom from Table 1. This can be viewed as the direct evidence to highlight the importance of the neutralization group. A different ability for the neutralization groups to lower the proton affinity of C5 atom could be found. For example, the inclusion of asparagine residue could only lower the PA of C5 atom about 19.8 kcal/mol (SCC-DFTB) and 17.3 kcal/mol (DFT), respectively. The resulted PA for C5 for this model (469.6 kcal/mol) is still much larger than tyrosine model (PA = 359.5 kcal/mol), which clearly suggests that only asparagine group as the neutralization function is not sufficient to activate the proton of C5 atom. Further addition of a positive imidazole group (model of ASN-GlcUA-HSP) significantly lowers the 10165



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Figure 2. Potential energy surface for the first step of β-elimination (A) and minimum energy path for the second step of protonation of the glcosydic bond (B) along the putative reaction coordinates calculated using the SCC-DFTB/MM method, in which d1 = dC5−H5 − dOH(Y408)−H5, d2 = dC4−O4, and d3 = dOH(Y408)−H5.

complex (EI) is found that the proton (H5) forms a stable covalent bond with OH atom of Y408. Snapshots for ES, TS1, and EI are plotted in Figure 3, and corresponding selected geometric parameters are listed in Table 2, in which the geometries for transition states are adopted using CPR calculations. As we can see, the first step of elimination seems to be a completely concerted process for the abstraction of proton H5 (HA1), the cleavage of C4−O4 glycosidic bond, and formation of unsaturated C4−C5 double bond. With proton (H5) transferring to OH (Y408) atom, the distance of H5−OH is shortened from 1.82 to 1.01 Å. Such distance means the formation of the OH bond and indicates that Y408 does have the function of general base. At the same time, the glycosidic bond is broken judged by the distance of C4−O4 elongated from 1.49 to 2.48 Å. In addition, with the formation of EI, the bond distance of C4−C5 is shortened from 1.52 to 1.34 Å, a typical distance for carbon−carbon double bond. Thus, the hybridization bond nature around C4−C5 bond along the reaction path deserves some discussion. Based on our NBO analyses using B3LYP/6-31G(d)/MM//SCC-DFTB/ MM single point calculations, the C4−C5 bond seems to stay between sp3 and sp2 hybridization before elimination: 2.54 2.42 ψNBO C4−C5 = 0.7129σsp (C4) + 0.7013σsp (C5), while the proton transfer (H5) to OH of Y408, and cleavage of glycosidic bond (O4−C4) transforms C4−C5 bond predominantly sp2 hybridNBO ization in the first transition state (TS1): ψC4−C5 = 0.7129σsp1.89(C4) + 0.7013σsp1.68(C5). Such a bond nature can prove that the formation of a carbon−carbon double bond. Once the cleavage of glycosidic bond and abstraction of H5 atom completed, a relatively strong hydrogen bond formed between glycosidic oxygen (O4) and potent proton donor (OH (Y408)) can be found with 1.80 Å for the distance of H5−O4. Meanwhile, the R462 provides additional stabilization to hold

Figure 3. Snapshots of the stationary points along the reaction path for the degradation of the hyaluronan tetrasaccharide molecule calculated with the SCC-DFTB/MM method. Carbon atoms are colored in green, blue for nitrogen atoms, and red for oxygen atoms.

the position of nonreducing saccharide unit. Such conformer represents a good geometry to fulfill the next proton transfer. Calculated energy barrier is about 1.3 kcal/mol, which is quite 10166



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Table 2. Selected Geometric Parameters for Stationary Points along the Hyaluronan Degradation Catalyzed by Wild-Type SpnHL at the SCC-DFTB/MM Level Theory distance (Å)

ES

TS1

EI

TS2

EP

H5(HA1)···OH(Y408) O4(HA1)···OH(Y408) H5(HA1)···C5(HA1) O4(HA1)···C4(HA1) H5(HA1)···O4(HA1) C5(HA1)···C4(HA1) C5(HA1)···C6(HA1) O62(HA1)···HH(Y408/H399) OH(Y408)···HH11(R462) OH(Y408)···HH21(R462) O4(HA1)···HH21(R462) O61(HA1)···HH21(N349) O3(HA1)···HE1(W291) O61(HA2)···HH11(R243) O62(HA2)···HH21(R243)

1.82 3.27 1.13 1.49 2.34 1.52 1.54 1.82 1.71 1.92 2.31 2.24 2.00 1.69 1.59

1.15 2.94 1.43 1.92 2.48 1.42 1.49 1.63 1.85 2.61 1.80 1.81 1.94 1.69 1.59

1.01 2.67 2.69 2.48 1.80 1.34 1.49 1.75 1.92 2.87 1.26 1.82 1.86 1.69 1.59

1.10 2.47 2.93 2.44 1.41 1.35 1.49 1.86 1.84 2.68 1.63 1.80 1.87 1.69 1.59

1.78 2.66 3.01 2.50 1.01 1.35 1.49 1.84 1.75 2.16 1.91 1.81 1.88 1.69 1.59

shallow. Obviously, the first step of β-elimination should be assigned to be the rate-determining step. Moreover, to stabilize the reaction intermediate species, hydrogen bond network formed between protein environment and substrates are well-maintained throughout the reaction, for example, the reducing HA unit hydrogen bonded by N349 and W291, while nonreducing part stabilized by R243. In fact, the function of the conserved R462 seems to deserve special interest. The mutation of corresponding arginine residue (labeling as R542) in Group B Streptococcal hyaluronate lyase was observed to significantly reduce the catalytic activity.56 First of all, the hydrogen bond formed between glycosidic oxygen (O4) and R462 represents the additional stabilization contribution provided by enzyme environment. On the other hand, throughout the reaction, a strong hydrogen bond formed between R462 and Y408 can be observed, which indicates that R462 could orientate the position of the general base. However, since R462 bears a positive charge on its side chain guanium group, which could weaken the basicity of hydroxide ion of Y408. Thus the function of the arginine residue is quite complicated. How to quantify the contribution of R462 to catalytic rate might be another interesting issue for further investigation. It has been suggested that the CPR could largely reproduce the experimental barrier height, especially those reactions involving proton transfer. We then include the CPR refined minimum energy path in Figure 4 for comparison. A stepwise reaction mechanism can be well established, although a shallow potential well can be located for the EI complex. Generally speaking, the first step for the β-elimination to generate the

unsaturated hexenuronic acid molecule is the rate-limiting step with a barrier height of 22.1 kcal/mol, which is consistent with the barrier height by MEP. The second step is related with the proton transfers from Y408 to glycosidic bond with a relatively low barrier of 1.44 kcal/mol. The overall reaction pathway is consistent with our SCC-DFTB/MM MEP calculations with no doubt. In all reactions involving catalysts, one important issue is how the catalyst is restored. In this work, it is then very interesting to combine our previous simulations of the generation of initial near attack conformer (NAC)57 via the substrate binding with current reactive processes. A complete enzyme catalysis process can be summarized in Scheme 3, in which we can see the Scheme 3. Proposed Catalytic Mechanism in the Degradation of Hyaluronan Tetrasaccharide Molecule Catalyzed by Hyaluronate Lyase from S. pneumonia

reaction starts from the Apo enzyme to the degradation of polysaccharide, and finally goes back to Apo enzyme to complete the catalytic cycle. Indeed, before substrate binding, the proton cannot spontaneously transfer from Y408 to H399 according to our former QM/MM simulation.11 When HA substrate binding, the Y408 changes to its ionized state very easily via the process of proton migration to H399. This process can lead to the formation of the so-called NAC. After a

Figure 4. CPR-refined energetic profiles using the SCC-DFTB/MM approach for the degradation of hyaluronan tetrasaccharide molecule. 10167



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lyase from S. pneumonia. Direct comparison with experimental data is thus not possible. 3.3. Truncated Active Site Model Calculations. To independently examine the viability of syn-elimination pathway for hyaluronan tetrasaccharide degradation using the SCCDFTB/MM method, a truncated active-site model at the B3LYP/6-31G(d,p) level of theory was carried out. The DFT investigation of the reaction pathway in the truncated active-site model identified five stationary points (ES, TS1, EI, TS2, and EP) and two corresponding transition states (TS1 and TS2) for the elimination and protonation steps, whose energies are listed in Table 3, and their selected key geometrical parameters and

stepwise PAD reaction, the glycosidic bond is broken to produce the unsaturated hexenuronic acid. At the same time, the positive charge is still maintained on H399. As we described in previous simulation,11 the proton migration from Y408 to H399 needs 3.0 kcal/mol if no any substrate molecule bound in the active site (Apo enzyme). On the contrary, the reverse proton transferring is very easy (0.23 kcal/mol). Such phenomenon could tell us one fact that after the degradation reaction is completed, the SphHL could restore its original status with the substrate molecule releasing to bulky solvent environment. Generally, the enzyme does not have any change after the catalytic reaction. 3.2. Potentials of Mean Force. Although MEP calculations can shed some lights on the catalytic mechanism for the SpnHL, the entropy effects were not included in the simulation. To account for dynamic effects caused by the enzyme and bulk solvent environment, we further carried out free energy simulations for the stepwise reaction along putative reaction coordinates. In the two-dimensional potential energy surface (Figure 2), since the energies in the upper left and lower right corner are rather high, those windows in these high energy regions were then omitted for saving computational cost. The calculated PMFs are depicted in Figure 5. Consistent with our reaction

Table 3. Energies of the Truncated Active-Site Model for the Hyaluronan Degradation Catalytized by Hyaluronate Lyase Calculated at B3LYP/6-31G(d,p) Level of Theory energy B3LYP/6-31G(d,p) B3LYP/6-31+G(d,p)// B3LYP/6-31G(d,p) free energy PCM (ε = 5) PCM (ε = 80)

ES

TS1

EI

TS2

EP

0.0 0.0

28.40 28.41

−8.20 −10.50

−6.31 −9.35

−10.87 −12.16

0.0 0.0 0.0

25.23 32.31 33.66

−10.40 0.10 3.34

−9.85 0.18 3.37

−11.60 −3.26 0.80

structures are displayed in Table 4 and Figure 6, respectively. Since the inclusion of the diffuse function does not qualitatively change the energies, we used the B3LYP/6-31G(d,p) results combined with the PCM calculations. Indeed, the inclusion of solvent effects could result in a nearly thermal neutral reaction, which is in good agreement with the CPR reaction pathway and PMF calculations. Although some minor differences could be found, the overall DFT calculated mechanism is still consistent with the QM/MM results. Particularly, the degradation of the substrate molecule is dominated by TS1, with a barrier height of 28.4 kcal/mol, which has a little higher with the addition of solvent contributions. It has to be noted that the truncated active site model is used to examine the qualitative reaction pathway. Due to the lack of the protein environment, quantitatively accurate results might not be expected for such a model. In fact, with the cleavage of glycosidic bond and proton migration to phenylhydroxide group, the bond distance of C4−C5 is shortened from 1.54 (ES) to 1.34 Å (EI), which agrees well with our QM/ MM simulation. Such a distance can ensure the formation of a double bond accordingly. Similarly, the EI is just metastable with a small (1.89 kcal/ mol) barrier toward EP. When solvent effects are included, this intermediate becomes either less stable (ε = 5) or even unstable (ε = 80). It should be pointed out that the transferring of proton H5 from Y408 to glycosidic O4 is associated with the second transition state, in complete agreement with the reaction path obtained with the SCC-DFTB/MM model. Indeed, the phenylhydroxide group in the DFT calculations does serve as the general base/general acid, which is the typical feature of syn-elimination mechanism as shown in Scheme 1. 3.4. Mutagenesis Study. To further understand the reaction processes and validate the SCC-DFTB/MM results, we have explored the impact of one of key active site residues, namely, N349. A mutant model was constructed with manually mutating N349 to A349. Such mutation could largely affect the neutralization of C5 carboxylate group, which has been recognized as one of the key issues for the enzymatic activity

Figure 5. Potentials of mean force for the first step of β-elimination (A) and the second step of protonation of the glcosydic bond (B) along the putative reaction coordinates calculated using SCC-DFTB/ MM method, in which d1 = dC5−H5 − dOH(Y408)−H5, d2 = dC4−O4, and d3 = dOH(Y408)−H5.

path calculations, only one transition state can be located for each step reaction. For the formation of intermediate, the calculated free energy barrier is about 21.5 kcal/mol, whereas it needs only about 0.75 kcal/mol to produce the product. Such low energy barrier indicates the intermediate complex is just metastable and kinetic insignificant. Clearly, the first step of βelimination can be assigned to be rate-limiting. Unfortunately, there is no direct rate constant reported for the hyaluronan 10168



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Table 4. Key Geometric Parameters of Stationary Points for Uncatalyzed Hyaluronan Degradation Calculated at the B3LYP/631G(d,p) Level of Theory distance (Å)

ES

TS1

EI

TS2

EP

H5(HA1)···Oη(Y408) O4(HA1)···Oη(Y408) H5(HA1)···C5(HA1) O4(HA1)···C4(HA1) H5(HA1)···O4(HA1) C5(HA1)···C4(HA1) C5(HA1)···C6(HA1) O62(HA1)···HH(Y408/H399) O62(HA1)···HH21(N349)

2.10 3.62 1.10 1.44 2.36 1.54 1.54 1.68 1.82

1.20 3.37 1.45 1.74 2.61 1.47 1.48 1.52 1.89

1.05 2.49 4.71 3.60 1.47 1.34 1.50 1.56 1.90

1.26 2.40 4.41 3.38 1.17 1.34 1.51 1.58 1.86

1.61 2.58 4.76 4.02 1.01 1.34 1.51 1.56 1.88

calculations, our simulations clearly suggest that N349 has some effects on the overall reactivity, but does not play the essential role, which is in agreement with experimental observations. Although our simulations indicate that N349A does weaken the overall catalytic activity, it still has to admit that N349 is not a perfect candidate to investigate the neutralization effects. In fact, in the gas phase proton affinity calculations, we built an interesting model in which the calcium ion replaces asparagine and positive histidine residues. The function of calcium ion to modulate the catalytic activity of HLs or other lyases has been discussed extensively.58,59 However, the reason for this is not well-known. In this work, as shown in Table 1, compared with the inclusion of Hsp, nearly another 100 kcal/mol reduction of PA for C5 atom can be observed to result in PA = 272.6 kcal/ mol. This obviously means more positive charge species close to the C5 carboxylate group could acidify H5 atom even more. In fact, it has been observed that, in some cases, Ca2+ is used to neutralize the C5 carboxylate group, for example, pectate lyase.4 Based on this observation, we might optimistically predict that the mutation of asparagine (N349) to lysine will increase the catalytic activity. Of course, this hypothesis clearly needs more experiments of site-directed mutagenesis and kinetic analysis to confirm.

for members in PL8. Indeed, in our QM/MM MD simulation, a relatively strong hydrogen bond was found to exist between the side chain of N349 and the C5-carboxylate group of the HA1 subunit. N349A mutation has been shown to weaken the activity according to some experimental kinetic data, about 6% activity against wild type SpnHL.10 In the simulation of N349A, the setup protocol and the definition of reaction coordinates for the reaction mechanism simulation are the same as we did in the wild type HL. The only difference is that 475 windows were employed in the PMF calculation for the first step of elimination in the mutagenesis simulations. The major function of N349 is suggested to form a hydrogen bond with the C5 carboxylate group of hyaluronic acid and then acidify the proton of H5. Indeed, in our PA calculations presented above, the addition of N349 could lower the proton affinity of C5 atom of GlcUA about 19.8 kcal/mol (SCCDFTB) and 17.3 kcal/mol (DFT), respectively. Although not significant, the decreasing of the proton affinity does indicate that the mutation of N349 could weaken the catalytic activity to some extent, which is consistent with experimental observations. Moreover, since the N349 does not directly participate in the degradation of hyaluronan, the overall reaction pathway in N349A can then be speculated to be the same as in wild type. First of all, a 2-D potential energy surface of the first step of βelimination, and the minimum energy path of the subsequent protonation of the glycosidic oxygen O4 are displayed in Figure S2. Snapshots for corresponding stationary states along the putative reaction coordinates are given in Figure S3, and some selected key geometric parameters are also included in Table S1 for comparison. Interestingly, the overall reactive processes in wild type and N349A are quite similar. The mutant does not change the assignment of the rate-limiting step, which is still the first step of generating the unsaturated hexenuronic acid with no doubt. We plotted the PMFs for these two steps in Figure 7. Similar with wild type SpnHL, only one transition state can be located to connect ES and EI with a free energy barrier height of 22.74 kcal/mol. The EI is metastable, since it only needs to overcome about 0.91 kcal/mol energy to produce the final neutral nonreducing sugar unit and restore the ionized Y408. Kinetic data analysis has shown that N349A has only about 6% activity against wild type enzyme,10 which corresponds about 1.67 kcal/ mol increasing in the activation energy according to transition state theory. In our computation, the value of ΔΔG between wild type and mutant enzyme is calculated to be 1.22 kcal/mol according to our simulations presented above. Clearly, our computational results are in consistent with the experimental observations and can offer additional support to the proposed mechanism. Therefore, in combination of PA and PMF

4. CONCLUSION To completely understand the detailed catalytic mechanism in enzyme systems, theoretical investigations based on QM/MM method have been proved to be very useful at the microscopic level. In this work, in combination with our previous work on the substrate binding features for the hyaluronan lyases from S. pneumonia, we proposed a complete catalytic cycle for the degradation of hyaluronan tetrasaccharide molecule. A synelimination mechanism for the degradation reaction catalyzed by SpnHL can be established by the hybrid QM/MM and DFT calculations, in which the ionized Y408 serves as the general base in the first step, while the general acid for the second step to produce the final product. Our simulation demonstrates that the first step of β-elimination is the rate-limiting step, while a relatively low barrier height can be found for the second step. Instead of the proposal of the H399 as the general base by Jedrzejas and co-workers, it is simulated to be positively charged and plays the major function of neutralizing the C5 carboxylate group. N349, on the other hand, can facilitate the reaction to some extent but is not critical as suggested by our mutagenesis simulation and proton affinity calculations. Based on our simulation, we further suggested that several mutants related with N349 or R462 might be helpful to facilitate the catalytic activity. Of course, more theoretical or experimental 10169



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Figure 7. Potentials of mean force for the first step of β-elimination (A) and the second step of protonation of the glcosydic bond (B) in N349A mutant along the putative reaction coordinates calculated using SCC-DFTB/MM method, in which d1 = dC5−H5 − dOH(Y408)−H5, d2 = dC4−O4, and d3 = dOH(Y408)−H5.

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ASSOCIATED CONTENT

S Supporting Information *

Full citation of refs 30 and 51; molecular orbitals for ES and TS1 using NBO analysis tool calculated at the B3LYP/MM// SCC-DFTB/MM level of theory; minimum energy pathways for the hyaluronan tetrasaccharide catalyzed by N349A, and corresponding geometry parameters and snapshots for all of stationary states at the SCC-DFTB/MM level of theory; Cartesian coordinates for the stationary states along the putative reaction coordinates for the truncated active site model optimized at the B3LYP/6-31G** level of theory. This material is available free of charge via the Internet at http:// pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; telephone number: 86-2884752094. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was funded by National Science Foundation of China (nos. 21073125 and 31170675) and by the Program for New Century Excellent Talents in University (no. NCET-100606). Parts of the results described in this paper are obtained on the Deepcomp7000 of Supercomputing Center, Computer Network Information Center of Chinese Academy of Sciences.

Figure 6. Stationary points along the reaction path for the degradation of hyaluronan tetrasaccharide molecule calculated with the B3LYP/631G(d,p) level of theory. Carbon atoms are colored in green, blue for nitrogen atoms, and red for oxygen atoms.

■

work can be devoted to get these issues clear. Finally, in combination of our previous QM/MM MD simulation of the formation of ES complex, we further proposed a complete catalytic cycle for the degradation of hyaluronan tetrasaccharide molecule catalyzed by SpnHL. It is our hope that our simulations could be useful in the development of antimicrobial pharmaceutical agents.

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Catalytic Mechanism of Hyaluronate Lyase from Spectrococcus

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