A Mechanistic Study of Trichoderma reesei Cel7B Catalyzed

Jul 3, 2013 - as suggested by several studies.4−6 The glycosyl bond cleavage is catalyzed by .... resuspended in 100 mM NaAc buffer (pH4.0), and lys...
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A Mechanistic Study of Trichoderma reesei Cel7B Catalyzed Glycosidic Bond Cleavage Yu Zhang, Shihai Yan,* and Lishan Yao* Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 266061, China S Supporting Information *

ABSTRACT: An ONIOM study is performed to illustrate the mechanism of Trichoderma reesei Cel7B catalyzed p-nitrophenyl lactoside hydrolysis. In both the glycosylation and deglycosylation steps, the reaction proceeds in a concerted way, meaning the nucleophilic attack and the glycosidic bond cleavage occur simultaneously. The glycosylation step is rate limiting with a barrier of 18.9 kcal/mol, comparable to the experimental value derived from the kcat measured in this work. The function of four residues R108, Y146, Y170, and D172, which form a hydrogen-bond network involving the substrate, is studied by conservative mutations. The mutants, including R108K, Y146F, Y170F, and D172N, decrease the enzyme activity by about 150−8000-fold. Molecular dynamics simulations show that the mutations disrupt the hydrogen-bond network, cause the substrate to deviate from active binding and hinder either the proton transfer from E201 to O4(+1) or the nucleophilic attack from E196 to C1(−1).

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reesei Cel7A, the major exoglucanase, with ∼0.9 Å Cα rootmean-square deviation (rmsd) for matched residues (∼45% sequence identity). One major difference between the two structures is that the endoglucanase has a ∼50 Å cleft but the exoglucanase has a tunnel with a similar length. The tunnel facilitates processive cellulose degradations while the cleft renders the enzyme ready to cleave cellulose randomly. Shortening the loops of T. reesei Cel7A that cover the tunnel increases the endo but decreases the exo activity,9 suggesting that different active-site structures serve different purposes. Though there has been no complex T. reesei Cel7B structure available, several T. reesei Cel7A structures9−12 imply the cleft is responsible for the cellulose chain binding and the glycosidic bond cleavage. Different binding properties of Cel7A and cel7B to cello-oligomer have been studied by molecular dynamics simulations.13 It is also suggested that the binding of T. reesei Cel7B to cellulose affects glucan clenching in the binding cleft.14 Same as T. reesei Cel7A, T. reesei Cel7B is a retaining glucosidase.15−17 It hydrolyzes glucan chains through a twostep mechanism, the glycosylation and deglycosylation steps. Computational studies18−20 of the T. reesei Cel7A catalyzed oligosaccharide hydrolysis showed that the glycosylation step involves a proton transfer from the general acid (E217) to the leaving group O4 and a nucleophile (E212) attack to the

INTRODUCTION Cellulose, the most abundant renewable biomass source on earth, is a polymer linked by unbranched β-1,4 glycosidic bonds. Cellulose can be hydrolyzed to glucose which can then be fermented to ethanol and other chemicals as substitutes for products derived from fossil fuels. Cellulases are attractive catalysts because they are more environmentally friendly than the strong acid catalysts. However, cellulases have limitations that the catalytic rate is slow, and high cellulase loads are needed. Researches on cellulases are actively pursued to understand the catalytic mechanism and enhance their efficiency. The filamentous fungus Trichoderma reesei (Hypocrea jecorina) is of particular interest due to its remarkable ability to secrete large amounts of cellulases that include exoglucanases cleaving cellobiose from cellulose strands ends, endoglucanases cleaving strands randomly, and β-glucosidases converting soluble cellodextrins and cellobiose to glucose. These cellulases usually consist of two domains, a large catalytic domain (CD) and a small carbohydrate-binding module (CBM), combined by an O-glycosylated linker peptide. The binding of the CBM of T. reesei cellulases to cellulose enhances the activity by increasing cellulases concentration on the cellulose surface.1−3 The CBM may also have a disruptive function against cellulose as suggested by several studies.4−6 The glycosyl bond cleavage is catalyzed by the CD domain. T. reesei Cel7B is one of the major endoglucanases that accounts for 5−10% of the total cellulases.7 The X-ray structure of apo T. reesei Cel7B CD has been determined,8 which shows a great similarity to that of T. © 2013 American Chemical Society

Received: April 22, 2013 Revised: June 8, 2013 Published: July 3, 2013 8714

dx.doi.org/10.1021/jp403999s | J. Phys. Chem. B 2013, 117, 8714−8722

The Journal of Physical Chemistry B

Article

The system was heated gently from 50 to 300 K in a 10 ps NVT simulation, after which a 20 ps NPT (1 atm, 300 K) MD simulation was performed. In both simulations, the heavy atoms were restrained. Then, a unrestrained NPT MD simulation was performed for 980 ps. Six MD snapshots, corresponding to the time points of 200, 400, 600, 800, 980, and 1000 ps, were used as the starting structures for NPT simulations with the length of 4 ns each. The R108K, Y146F, Y170F, and D172N mutant simulations were run in the same way. In the protein CA and substrate rmsd calculation, the last structure from 5000 unrestrained energy minimizations (following the restrained minimization mentioned above) was selected as the reference. ONIOM Calculation of the WT. The average structure of the WT was calculated from six 4 ns trajectories. The snapshot with the structure closest to the average was selected as the initial model for QM/MM calculations, which were carried out at ONIOM(B3PW91/6-31+G(d,p):AM1)44−47 level and performed with Gaussian 09 program.48 The model system includes all the residues whose atoms are within 10 Å from the O4(−1) of PNPL ligand, corresponding to a total of 36 residues (Figure 1). All MD water molecules were excluded.

anomeric carbon atom C1, whereas the deglycosylation involves a proton transfer from a water molecule to E217 and a hydroxide nucleophilic attack to C1. The importance of the two catalytic residues is validated by the mutagenesis study that E212Q and E217Q mutants decrease the T. reesei Cel7A hydrolysis activity toward 2-chloro-4-nitrophenyl β-lactoside by 85- and 370-fold, respectively.11 A third acidic residue D214 is thought to position E212 ready for the nucleophilic attack. The sequence alignment shows that the corresponding general acid and nucleophile in T. reesei Cel7B are E201 and E196, and the assisting acidic residue is D198, all located in the cleft.8 Besides these three residues, there are several other conserved ones in the active site, especially in the −1 and −2 binding subsite. The T. reesei Cel7A cello-oligosaccharide complex structures9−12 reveal that several but not all the residues interact with the ligand directly. What are the roles of these residues? How do they contribute to the catalysis? In this work, we adopt the ONIOM21−23 and molecular dynamics simulation methods in combination with the steady-state enzyme kinetics experiments to study the mechanism of T. reesei Cel7B catalyzed pnitrophenyl lactoside (PNPL) hydrolysis. The ONIOM method has been widely used to study enzymatic catalysis mechanisms; e.g.,24−30 the MD simulation method has also been successfully employed to study cellulases, including Cel7A, Cel7B cellulose binding,14,31 Cel6A cello-oligomer interaction,32 cellobiose release from Cel7A,33 etc. Our study shows that similar to T. reesei Cel7A,18−20 a concerted mechanism is adopted by Cel7B in both the glycosylation and deglycosylation steps. Then, several activesite mutants in −1 and −2 binding subsites are studied. A large variation of the catalytic activity decease is found for different mutants from the steady-state kinetics study, highlighting the importance of these residues. MD simulations of the mutants show that though these residues do not involve in the catalysis directly, they facilitate the reaction by restraining the substrate for the proton transfer and nucleophilic attack.

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METHODS AND MATERIALS MD Simulation. The apo form T. reesei Cel7B crystal structure (1EG1) reported by Jones8 was used as a starting structure with the protonation states determined by a pKa analysis.34−37 The ligand PNPL was added in the following way. 1EG1 was fitted to 8CEL, the T. reesei Cel7A cello-oligomer complex crystal structure, by using PyMOL (Schrödinger, LLC). The cello-oligomer coordinates were transferred to 1EG1 and then the ligand PNPL was inserted by fitting the lactoside moiety to the glycosyl located at −2 and −1 subsites and placing the p-nitrophenyl moiety at +1 subsite. The cellooligomer was then removed from the 1EG1 active site. The simulations were carried out with the AMBER 11 molecular dynamics package.38 The Amber force fields of FF99SB39 and GAFF40 were employed to model the enzyme and the ligands. The Antechamber module of Amber 11 was employed to calculate BCC charges41,42 for the optimized PNPL structure with AM1 Hamiltonian. Six Na+ ions were employed to neutralize the system using the Amber LEAP tool. The complex structure was immersed in an octahedral box of explicit TIP3P43 water molecules, with a 12.0 Å distance between the solvent box wall and the nearest solute atoms. Totally, there were about 50 000 atoms in the system. The details of MD simulations are as follows. Initially, the system was minimized for 5000 steps with all heavy atoms restrained by the harmonic potential (k = 10 kcal/(mol·Å2)).

Figure 1. ONIOM model of the WT Cel7B ES state. A total of 36 residues are included in the model. The residues in the high level, including E201, E196, and PNPL, are depicted in balls and lines while those in the low level are represented with lines.

The system was divided into two layers, as implemented in the ONIOM method.49 The active-site amino acids, E201 and E196 (modeled by propionic acid), as well as the moiety of PNPL ligand located at −1 and +1 subsites were included in the high layer and treated with the quantum mechanical method. All remainder were included in the low layer treated with AM1. The substrate complex was optimized with all the atoms in the high layer as well as glycosyl at −2 subsite, D198 and N142 from the lower layer allowed relaxing. The optimized substrate complex was used as the starting structure for the subsequent study. For the glycosylation step, a 2D energy surface was constructed by scanning C1(−1)···Oε2(E196) and C1(−1)···O4(+1) distances independently from 1.46 to 3.16 Å. For the deglycosylation step, C 1(−1)···O ε2 (E196) and 8715

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Table 1. Primary Hydrogen Bond Distancesa (Å) in the Active Site of WT and Mutants donor···acceptor Nη2(R108)···O3(−2) Nη2(R108)···O4(−2) Oη(Y146)···O2(−2) Oγ(S318)···O2(−2) O3(−1)···Oδ1(D172) O3(−1)···Oδ2(D172) O2(−1)···Oε1(E196) O6(−1)···Oδ1(D198) Nη1(R108)···Oδ1(D35) Oη(Y146)···Oη(Y170) Oη(Y170)···Oδ2(D172) Oδ2(D198)···Oε2(E196) a

WT 3.07 3.19 2.80 2.90 3.28 3.38 2.68 3.09 2.86 2.78 3.66 2.58

± ± ± ± ± ± ± ± ± ± ± ±

0.30 0.29 0.14 0.21 0.70 0.68 0.16 0.79 0.19 0.12 1.07 0.09

R108K 4.78 5.12 2.83 2.86 3.07 3.08 2.61 5.49 3.68 2.82 2.83 2.58

± ± ± ± ± ± ± ± ± ± ± ±

Y146F 3.45 ± 3.70 ± N/A 3.42 ± 3.73 ± 3.78 ± 3.32 ± 3.68 ± 3.11 ± N/A 2.91 ± 2.62 ±

0.74 0.91 0.16 0.19 0.48 0.32 0.10 1.31 1.06 0.14 0.34 0.09

Y170F

0.84 0.67 0.71 1.66 1.58 1.59 1.25 0.48 0.34 0.28

4.63 ± 3.08 ± 4.27 ± 3.85 ± 4.49 ± 4.92 ± 3.01 ± 6.20 ± 3.46 ± N/A N/A 3.11 ±

0.54 0.35 0.44 0.80 0.91 0.75 0.35 0.90 0.75

0.91

D172N 3.04 3.15 2.82 2.89 4.66 3.52 3.77 3.17 2.95 2.83 4.44 2.60

± ± ± ± ± ± ± ± ± ± ± ±

0.27 0.25 0.14 0.19 0.73 0.62 0.77 0.82 0.29 0.15 0.49 0.10

The distance was averaged over six 4 ns MD trajectories.

substrate (final concentration 1, 1.8, 2.5, 3.5, 5, 6.5, 8 mM) in 100 mM acetate buffer (pH 4.0) with 0.3 μM WT Cel7B-CD. Aliquots of 10 μL were taken at time intervals (0, 5, 8, 11, 14, and 17 min) during the incubation and transferred immediately to a microplate containing 190 μL of 1 M Na2CO3 to stop the reaction. The amount of pNP liberated was determined by measuring the absorbance at 405 nm using a microplate reader (Ultrospec Visible plate reader II 96, GE Healthcare BioScience). The standard Michaelis−Menten equation was applied to fit the kinetics kcat and Km values using an inhouse script. The amount of the added enzyme and the sampling time were adjusted accordingly for the mutants to reliably determine the reaction rates. All the experiments were measured in triplicate.

C1(−1)···O(water) coordinates were selected and scanned from 1.46 to 3.16 Å. The scanning distance interval was 0.10 Å. To consider the effect of other residues in the vicinity of the active site, the obtained stationary states from distance scanning were reoptimized with more residues including R108, N142, S144, Y146, S148, Y170, D172, Q174, and S318 unrestrained. The solvation effect was included (at ONIOM(B3PW91/631+G(d,p):AM1) level) using the self-consistent reaction field (SCRF) method employing the polarizable continuum model (PCM)50,51 with a dielectric constant of 78.5 while the structures of ES, TS1, EI(EI′), and EP were taken from ONIOM(B3PW91/6-31+G(d,p):AM1) gas-phase optimizations. The effect of different density functionals was also considered by optimizing the structures using B3LYP//631+G(d,p) for the high level while retaining AM1 for the low level. Cloning, Expression, and Purification. The DNA encoding residues of the Cel7B catalytic domain (Cel7B-CD) from T. reesei QM9414 and a 6*His tag at the C-terminus was ligated with the vector pET-20b, which was digested with the restriction enzymes, NdeI and HindIII. The ligation mixture was transformed into an E. coli strain DH10B. The correct coding sequence of the cloned catalytic domain of Cel7B gene was verified by DNA sequencing. The expression vector (pET20b-Cel7B-CD) was then transformed into E. coli strain origami (DE3). All the mutations were made by PCR-based site-directed mutagenesis and verified by DNA sequencing. All the mutants were expressed and purified in a similar way. Briefly, 250 mL of LB medium containing 100 μg/mL ampicillin was inoculated with a fresh colony of expression strain origami (DE3) containing pET-20b-Cel7B-CD. The culture was grown at 37 °C with vigorous shaking (∼200 rpm). When the OD600 of the culture reached 0.8−1.2, a final concentration of 1 mM of IPTG was added to induce the expression of the protein at 16 °C and for 48 h. The cells were harvested by centrifugation, washed twice with water, resuspended in 100 mM NaAc buffer (pH4.0), and lysed by ultrasound sonication. The lysed cells were centrifuged (9600g, 4 °C, 20 min) and the resulting supernatants were purified by Ni-NTA affinity chromatography (Novagen). The purity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentration was measured by UV spectroscopy using an extinction coefficient of 60 380 M−1 cm−1 at 280 nm.52 Kinetic Studies. Incubations of Lac-β-pNP as substrates were carried out at 30 °C using a mixture (100 μL) containing

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RESULTS AND DISCUSSION MD of the WT Cel7B. The Cα rmsd of the protein obtained from six 4 ns classical MD simulations, ranging around 1.6 Å, illustrates the enzyme is fully equilibrated (Figure S1 in the Supporting Information). The interactions between the ligand PNPL and the protein are listed in Table 1. A total of eight hydrogen bonds are formed between PNPL and the active site residues, among which H-bonds OηH(Y146)···O2(−2, from subsite −2), O2H(−1)···Oε1(E196), and OγH(S318)···O2(−2) are relatively strong. Four weak hydrogen bonds were found, with two between O3H(−2) and Oδ1,δ2(D172), and the other two between Nη2H(R108)···O3,4(−2). Residues, including R108, Y146, E196, and D172, are H-bonded with the substrate, as well as other residues. For example, OηH(Y146) forms a Hbond with Oη(Y170) which is H-bonded as a donor to Oδ(D172). All hydrogen bonds with PNPL are located in −2 and −1 subsites, suggesting the glycosyl group is well restrained by the hydrogen-bond network (Figure 2) but the pnitrophenyl group is rather mobile. The dihedral angle C2− C1−O5−C5 of the sugar ring at −1 subsite displays a two-state behavior. The major state, with the occurrence frequency of 95.5%, has the average of −42.7 ± 8.9°, corresponding to the 4 C1 chair conformation. The minor state has the average of 58.7 ± 11.1°, corresponding to the 1,4B boat conformation. Catalytic Mechanism from ONIOM Calculations. For a retaining GH enzyme such as T. Reesei Cel7B, the catalysis involves two steps, glycosylation and deglycosylation. Two distances C1(−1)···Oε2(E196) and C1(−1)···O4(+1) were selected to describe the glycosylation step. The shortening of C1(−1)···Oε2(E196) distance corresponds to the nucleophilic attack from E196 to C1(−1) and the lengthening of 8716

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C1(−1)···O(water) distances. The former describes the glycosidic bond between ligand and protein whereas the latter is an indicator for the nucleophilic attack from water. The starting structure of the deglycosylation (defined as EI′) resembles the EI state of the glycosylation step except that the leaving group of p-nitrophenol is substituted by a water molecule. Considering Km for PNPL is 14 mM, corresponding to the binding energy of 2.5 kcal/mol for the substrate and the main contribution is from the lactoside group due to the hydrogen-bond interactions, the binding of p-nitrophenol should be very weak. In other words, the energy difference between EI′ and EI is very small, which is neglected in this work. An early study by Bu et al.33 also showed that the cellobiose release from Cel7B active site has a barrier of 5 kcal/ mol. So the release of p-nitrophenol, which is smaller and forms fewer interactions with Cel7B, is facile. The water molecules would fill in the space left by p-nitrophenol. After manually adding one water molecule approaching the C1(−1) site, the system was optimized. A hydrogen bond is formed between the water molecule and E201 side-chain carboxyl group (EI′, Figure 4). Two energy minima are identified in Figure 5, with one corresponding to EI′ and the other being the enzyme product state (EP). EP, located on the lower right corner, has the energy lower than that of the EI′ state by 13.9 kcal/mol. A barrier of 10.5 kcal/mol is found along the MEP at the C1(−1)···Oε2(E196) distance of 2.66 Å and the C1(−1)···O(water) distance of 2.16 Å. Figure 5 indicates that the transition state geometrically resembles the product, predicting a late transition state (TS2). Similar to the glycosylation step, a concerted mechanism is adopted in the deglycosylation reaction. The energies for all the stationary states are listed in Table 2 and the structures are shown in Figure S2 in the Supporting Information. In the structure optimizations, the atoms in the high level and several atoms in the low level were allowed to move while the rest of the system was fixed. To investigate whether the relaxation of other residues in the vicinity of the active site has a dramatic effect on the relative energies, we reoptimized all the stationary states by allowing nine more residues close to the active center to move (Methods and Materials). The relative energies, listed in Table 2, show an energy increase of 2−3 kcal/mol for TS1, EI, and TS2. Using B3LYP for the high-level DFT has a minor effect on the relative energies. Adding the solvation effect decreases the TS1 and EI (EI′) energies by 2.3 and 6.7 kcal/mol, respectively. Thus, allowing more residues to relax and including the solvation term have some effects on the relative energies but do not change the overall energy landscape dramatically. The overall rate-limiting step is the glycosylation which has a barrier of 18.9 kcal/mol, comparable to the predicted free energy barrier (17.5 kcal/mol) of T. reesi Cel7A catalyzed cello-oligomer hydrolysis.18 Itinerary of Sugar Ring at the −1 Subsite. The sugar ring at the −1 subsite of PNPL has the 4C1 chair conformation in the ES state with the C2−C1−O5−C5 dihedral angle of −54.8°. This sugar ring puckers along the glycosylation, via the 4 E geometry, into the 4H3 half-chair structure at the TS1 transition state with the C2−C1−O5−C5 dihedral of −6.7°. When the glycosylation reaction is finished, the 4C1 chair structure is recovered and the C2−C1−O5−C5 dihedral decreases to −58.6° at the EI state. The C2−C1−O5−C5 dihedral changes slightly to −52.3° when a water molecule substitutes the p-nitrophenol (EI′). In the step from EI′ to TS2,

Figure 2. H-bond network around the substrate PNPL. The H-bonds are drawn with dashed lines.

C1(−1)···O4(+1) distance describes the glycosidic bond cleavage. Figure 3 presents the contour plot of the potential

Figure 3. Contour plot of the potential energy surface for the glycosylation step obtained at ONIOM (B3PW91/6-31+G(d,p):AM1) level. The x-axis is the C1(−1)···Oε2(E196) distance, the reaction coordinate for nucleophilic attack. The y-axis is the C1(−1)···O4(+1) distance, describing the glycosidic bond cleavage or formation. The reaction path from the reactant to the glycosylated intermediate is shown with a green dashed line, with a barrier of 18.9 kcal/mol.

energy surface (PES) for this step. For the enzyme substrate state (ES, Figure 4), C1(−1) and O4(+1) are covalently bonded, while the Oε2(E196) is hydrogen bonded with the C1H(−1) group. This state corresponds to the energy minimum on the lower right corner of Figure 3. The other energy-minimum state, on the top left corner, denotes the glycosylated intermediate (EI, Figure 4) with a new covalent C1(−1)−Oε2(E196) bond formed (the bond length of 1.465 Å). For EI, the C1(−1)−O4(+1) bond is cleaved, and the proton is transferred from E201 to O4(+1) of PNPL. Meanwhile, a strong hydrogen bond is formed between E201 and PNPL (Oε2(E201)···O4(+1) distance of 2.618 Å). The relative energy of EI is almost equal to that of ES. The minimum-energy path (MEP), shown with a dashed line in Figure 3, connects two minima of ES and EI. The transition state (TS1, Figure 4) locates between ES and EI states along the MEP. For this transition state, the C1(−1)···Oε2(E196) and C1(−1)···O4(+1) distances are 2.56 and 2.06 Å, respectively. Thus, a concerted mechanism is adopted by the enzyme for the glycosylation step. The contour plot of the PES for the deglycosylation step is shown in Figure 5, as a function of C1(−1)···Oε2(E196) and 8717

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Figure 4. Schematic representation of different states along the Cel7B-catalyzed PNPL hydrolysis.

Table 2. Relative Energies of Stationary States Obtained at Different Levels B3PW91a B3PW91(Relx)b B3LYP(Relx)b B3PW91(Solv)c

ES

TS1

EI (EI′)

TS2

EP

0.0 0.0 0.0 0.0

19.0 22.2 21.1 18.9

0.0 2.9 4.0 −3.8

10.5 12.8 12.1 11.7

−13.9 −14.9 −13.9 −14.7

a

The relative energies were obtained at ONIOM(B3PW91/631+G(d,p):AM1) level with the atoms in the DFT layer, −2 subsite glycosyl group, residues D198 and N142 from the AM1 layer allowed to move while the rest of the system was restrained. bONIOM(B3PW91/6-31+G(d,p):AM1) and ONIOM(B3LYP/6-31+G(d,p):AM1) were adopted for the energy calculations while allowing more residues in the vicinity of the active site to relax (see main text). c PCM solvation model was incorporated to study the solvation effect. Figure 5. Contour plot of the potential energy surface for the deglycosylation step obtained at ONIOM (B3PW91/6-31+G(d,p):AM1) level. The x-axis is the C1(−1)···Oε2(E196) distance, describing the glycosidic bond between the intermediate and the protein. The y-axis is the C1(−1)···O(W) distance where O(W) is the oxygen atom of a water molecule, describing the nucleophilic attack. The reaction path from the glycosylated intermediate to the product is shown using a green dashed line, with a barrier of 10.5 kcal/mol.

H-bonded to the ligand. A total of nine enzymes from glycoside hydrolase family 7 (GH7) have their structures solved by the Xray crystallography (http://www.cazy.org/GH7_structure. html), including Cel7A from Trichoderma harzianum,53 T. reesei,11 Rasamsonia emersonii,54 and Heterobasidion annosum,55 Cel7B from Melanocarpus albomyces,56 T. reesei,8 Fusarium oxysporum,57 and Humicola insolens,58 and Cel7D from Phanerochaete chrysosporium.59 All structures share a similar fold and active-site interactions. The sequence alignment of these glycoside hydrolases (using ClustalW60) shows that the active site has a conserved catalytic motif E-X-D-X-X-E, with the first E as the nucleophile, the second E as the general acid, and the D as the assisting acid. The alignment of the nine sequences shows that various residues are strictly conserved in the active site, including the catalytic acid/base E201, the nucleophile E196, and several other residues in the −2, −1, and +1 binding site in close contact with the ligand such as R108, N142, Y146, Y170, D172, Q174, D198, H212, S318, and

the conformation of −1 subsite ring changes from 4C1 to 4H3 half-chair with the C2−C1−O5−C5 dihedral angle changing from 60° to 3.5°. From TS2 to the product state, 4H3 changes via E3 back to the 4C1 conformer with the C2−C1−O5−C5 dihedral of −59.7°. Selection of Mutants. As revealed from the ONIOM calculations, the general acid/base E201 and nucleophile E196 are essential for the catalysis. But the roles of other residues in the active site are not well understood, including those directly 8718

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binding of PNPL is weaker. The catalytic rate kcat of 525.7 min−1 corresponds to a barrier of 16.1 kcal/mol, as calculated from the transition-state theory, which is comparable to that from the ONIOM calculation. R108K decreases the kcat/Km by about 130-fold and the main contribution is from kcat. In comparison, Y146F and D172N reduce the activity by ∼1100fold and Y170F reduces it by ∼7700-fold. All the mutations are rather conservative and only modify the residues slightly, but cause a dramatic decrease of catalytic efficiency. MD Simulation of the Mutants. To understand why mutations cause such a drastic change of activity, a series of MD simulations was performed for R108K, Y146F, Y170F, and D127N in complex with the substrate PNPL. Overall, all-Cα rmsd from the X-ray structure is similar for each enzyme substrate complex (Figure S1 in the Supporting Information). And the Cα root-mean-square fluctuation (rmsf) is also quite similar (Figure S3 in the Supporting Information), suggesting the mutations have a very minor effect on the overall structure and dynamics. Further trajectory analyses suggest the active-site interactions are modified by mutants (Figures S4 and S5 in the Supporting Information). R108K. Considering the side chain of R108 is about 10 Å away from the C1(−1)···O4(+1) bond where the catalysis occurs, it is surprising that R108K causes a dramatic change of catalytic efficiency. In the simulation of WT Cel7B, R108 forms two weak hydrogen bonds with O3(−2) and O4(−2) through Nη2H, and a strong H-bond with Oδ1(D35) (Table 1). All three hydrogen bonds disappear in the R108K mutant. The mutant also disrupts the weak hydrogen bond between O6H(−1) and Oδ1(D198) (Table 1). To gain more insight about the change, two geometric parameters, r1, the C1(−1)···Oε2(E196) distance and r2, the O4(+1)···Oε2(E201) distance, were monitored. The former reflects the feasibility of the nucleophilic attack while the latter is directly related to the proton transfer. For the WT Cel7B, the distribution of (r1, r2) is shown in Figure 6, with an

W320. The interactions between the protein and the ligand are mainly through the residue side chains. R108 and S318 are hydrogen bonded to O3(−2); Y146 is H-bonded to O2(−2); N142 is H-bonded to O6(−1); D172 is H-bonded to O3(−1); W320 is in close contact with the −1 sugar ring through the van der Waals interaction. Y170 has no direct contact with the substrate but bridges Y146 and D172 through a hydrogen-bond network. The sites selected for further characterization are R108, Y146, Y170, and D172. The catalytic residues E196 and E201, as well as the assisting residue D198, were not chosen since they have been studied elsewhere in the T. reesei Cel7A system.11 Conservative single mutations R108K, Y146F, Y170F, and D172N, which were selected to minimize the structural perturbation, were characterized experimentally and computationally. The results are discussed below. Kinetic Rates. The initial reaction rate was measured at different substrate concentrations and fitted to the Michaelis− Menten equation (Table 3). The WT Cel7B has a kcat of 525.7 Table 3. Kinetic Constants for the Hydrolysis of PNPL by the WT Cel7B and Mutants

WT R108K Y146F Y170F D172N a

Km (mM)

kcat (min−1)

14.3 ± 17.1 ± 13.1 ± N/A 21.1 ±

525.7 ± 48.3 4.77 ± 0.99 0.403 ± 0.08 N/A 0.71 ± 0.14

1.8 4.6 3.3 4.7

kcat/Km (min−1mM−1)

rel activitya

± ± ± ± ±

1 0.00766 0.00084 0.00013 0.00091

36.8 0.279 0.0308 0.0047 0.0337

1.3 0.017 0.0016 0.0004 0.0009

Relative activity is defined as the ratio (kcat/Km)mut/(kcat/Km)WT.

min−1, similar (MeUmbL) and which implies a these substrates. larger than that

to that of 4-methylumbelliferyl lactoside cellotriose catalyzed by the same enzyme,52,61 similar catalytic mechanism is employed for But the Km value of PNPL is about 10 times for MeUmbL and cellotriose, suggesting the

Figure 6. Scattering plots for the distances of r1 and r2. r1 and r2 denote the distance of C1(−1)···Oε2(E196) and Oε2(E201)···O4 (+1), respectively. Each plot includes 2400 dots extracted from the MD snapshots, corresponding to six 4 ns trajectories. 8719

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in this mutant, consistent with the experimental findings that Y170F has the lowest activity. D172N. The carboxyl group of D172 is H-bonded with O3(−1) and the hydroxyl of Y170 in the WT form. Mutating D172 to an asparagine largely disrupts these two hydrogen bonds. But the amine group of N172 forms a new hydrogen bond with Oε1 of E196, the nucleophile, with the average heavy atoms distance of 2.82 ± 0.25 Å. This H-bond pulls E196 slightly away from C1(−1), the nucleophilic attack site, revealed by the average r2 distance of 3.41 Å which is ∼0.2 Å longer than that of WT. It is expected that the hydrogen bond between N172 and E196 pulls the electron density away from E196 carboxyl group so that Oε2(E196) becomes less negative charged. These changes make the nucleophilic attack more difficult for the mutant.

average of (3.22 Å, 3.47 Å). r1 is more symmetric and has a much smaller standard deviation (0.16 Å) than that of r2 (0.79 Å). The r2 distribution has a positive skew. The skewed distribution is corroborated with the distribution of the substrate rmsd from the optimized docking model (Figure 7),

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CONCLUSION A catalytic mechanism is proposed for Cel7B-catalyzed PNPL hydrolysis based on the ONIOM calculation. The reaction proceeds through glycosylation and deglycosylation steps. In the glycosylation step, along with the transfer of a proton from E201 to O4(+1), the side-chain carboxyl of E196 attacks and forms a covalent bond with C1(−1) and the glycosidic bond C1(−1)−O4(+1) is cleaved. One product, p-nitrophenol, is released. In the deglycosylation step, the hydroxide of a water molecule attacks and bonds with C1(−1) and simultaneously breaks the covalent bond between C1(−1) and Oε2(E196) whereas the proton of the water transfers to E201. The other product lactose is formed and the enzyme is regenerated for a new catalytic cycle. The concerted mechanism is adopted in both steps with the glycosylation step being rate-limiting. Four conservative mutants including R108K, Y146F, Y170F, and D172N display a low catalytic activity, in the order of R108K > D172N > Y146F > Y170F. MD simulations show that the mutants maintain the overall protein structure and dynamics but alter the interactions in the active site. The measured activity loss of the mutants is explained qualitatively by larger C1(−1)···Oε2(E196) or O4(+1)···Oε2(E201) distances in the simulation, suggesting the mutants either hinder the proton transfer from E201 to O4(+1) or the nucleophilic attack from E196 to C1(−1).

Figure 7. Histogram of the substrate PNPL rmsd from the reference structure. Each histogram is built with a bin size of 0.2 Å, from 2400 MD snapshots (24 ns).

suggesting the skewness of r2 distribution is caused by the substrate’s deviation from the starting structure. For the R108K mutant, the average (r1, r2) values are (3.15 Å, 4.09 Å). The average O4(+1)···Oε2(E201) distance is considerably larger than the WT simulation, suggesting the proton transfer is more difficult in the R108K mutant. Correspondingly, a larger substrate RMSD is also observed for the mutant than the WT (Figure 7). Y146F. In the WT simulation, the hydroxyl group of Y146 is H-bonded to O2(−2) and Y170. Removing this hydroxyl group has a profound effect, not just limited to the two hydrogen bonds. The MD trajectories of the mutant show most of the hydrogen bonds in the active site are weakened or disrupted (Table 1). The substrate average rmsd from the starting structure is 1.83 ± 0.79 Å, considerably larger than that of WT, suggesting in part of the MD trajectories the substrate drifts away from the active site (Figure 7). This is reflected by the (r1, r2) distribution as well (Figure 6). The average r1 and r2 values are 3.58 and 3.81 Å, respectively. Both are larger than the WT, suggesting the proton transfer and nucleophilic attack are more difficult in the mutant. Y170F. In the WT enzyme−substrate complex, Y170 does not interact with the substrate directly. Similar to Y146F mutant, most of the hydrogen bonds listed in Table 1 are disrupted. The substrate displays a much larger rmsd from the starting structure than WT (Figure 7) and the average r1 and r2 distances are 3.92 and 6.24 Å, respectively, the largest among all the enzyme−substrate complexes. This observation suggests the proton transfer and nucleophilic attack are the most difficult

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ASSOCIATED CONTENT

S Supporting Information *

The protein and ligand rmsds as well as the protein rmsfs of the WT and mutants, Gaussian-optimized geometries of the WT at various states, mutant structures of R108K, Y146F, Y170F, and D172N, and the complete lists of refs 9, 31, 38, 48, and 60. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (S.Y.); [email protected] (L.Y.). Phone: 86 532 80662791 (S.Y.); 86 532 80662792 (L.Y.). Fax: 86 532 80662778 (S.Y.); 86 532 80662778 (L.Y.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are thankful to Supercomputing Center of Chinese Academy of Sciences (CAS) for providing the computer 8720

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(15) Claeyssens, M.; Tomme, P.; Brewer, C. F.; Hehre, E. J. Stereochemical Course Of Hydrolysis And Hydration Reactions Catalyzed By Cellobiohydrolase-I And Cellobiohydrolase-Ii From Trichoderma-Reesei. FEBS Lett. 1990, 263 (1), 89−92. (16) Knowles, J. K. C.; Lentovaara, P.; Murray, M.; Sinnott, M. L. Stereochemical Course Of The Action Of The Cellobioside Hydrolase-I And Hydrolase-Ii Of Trichoderma-Reesei. J. Chem. Soc., Chem. Commun. 1988, 21, 1401−1402. (17) Claeyssens, M.; Henrissat, B. Specificity Mapping Of Cellulolytic Enzymes - Classification Into Families Of Structurally Related Proteins Confirmed By Biochemical-Analysis. Protein Sci. 1992, 1 (10), 1293−1297. (18) Barnett, C. B.; Wilkinson, K. A.; Naidoo, K. J. Molecular Details From Computational Reaction Dynamics For The Cellobiohydrolase I Glycosylation Reaction. J. Am. Chem. Soc. 2011, 133 (48), 19474− 19482. (19) Li, J. H.; Du, L. K.; Wang, L. S. Glycosidic-Bond Hydrolysis Mechanism Catalyzed By Cellulase Cel7a From Trichoderma Reesei: A Comprehensive Theoretical Study By Performing Md, Qm, And Qm/Mm Calculations. J. Phys. Chem. B 2010, 114 (46), 15261− 15268. (20) Yan, S. H.; Li, T.; Yao, L. S. Mutational Effects On The Catalytic Mechanism Of Cellobiohydrolase I From Trichoderma Reesei. J. Phys. Chem. B 2011, 115 (17), 4982−4989. (21) Vreven, T.; Morokuma, K.; Farkas, O.; Schlegel, H. B.; Frisch, M. J. Geometry Optimization With Qm/Mm, Oniom, And Other Combined Methods. I. Microiterations And Constraints. J. Comput. Chem. 2003, 24 (6), 760−769. (22) Lundberg, M.; Kawatsu, T.; Vreven, T.; Frisch, M. J.; Morokuma, K. Transition States In A Protein Environment - Oniom Qm:Mm Modeling Of Isopenicillin N Synthesis. J. Chem. Theory Comput. 2009, 5 (1), 222−234. (23) Chung, L. W.; Li, X.; Sugimoto, H.; Shiro, Y.; Morokuma, K. Oniom Study On A Missing Piece In Our Understanding Of Heme Chemistry: Bacterial Tryptophan 2,3-Dioxygenase With Dual Oxidants. J. Am. Chem. Soc. 2010, 132 (34), 11993−12005. (24) Ding, L. N.; Chung, L. W.; Morokuma, K. Reaction Mechanism Of Photoinduced Decarboxylation Of The Photoactivatable Green Fluorescent Protein: An Oniom(Qm:Mm) Study. J. Phys. Chem. B 2013, 117 (4), 1075−1084. (25) Wang, X. Q.; Hirao, H. Oniom (Dft:Mm) Study Of The Catalytic Mechanism Of Myo-Inositol Monophosphatase: Essential Role Of Water In Enzyme Catalysis In The Two-Metal Mechanism. J. Phys. Chem. B 2013, 117 (3), 833−842. (26) Chung, L. W.; Hirao, H.; Li, X.; Morokuma, K. The Oniom Method: Its Foundation And Applications To Metalloenzymes And Photobiology. Wiley Interdiscip. Rev.: Comput. Mol. Sci. 2012, 2 (2), 327−350. (27) Ma, Y. Y.; Sun, Q.; Li, Z.; Yu, J. G.; Smith, S. C. Theoretical Studies Of Chromophore Maturation In The Wild-Type Green Fluorescent Protein: Oniom(Dft:Mm) Investigation Of The Mechanism Of Cyclization. J. Phys. Chem. B 2012, 116 (4), 1426−1436. (28) Hirao, H.; Morokuma, K. Oniom(Dft:Mm) Study Of 2Hydroxyethylphosphonate Dioxygenase: What Determines The Destinies Of Different Substrates? J. Am. Chem. Soc. 2011, 133 (37), 14550−14553. (29) Wang, X.; Hirao, H. Oniom (Dft:Mm) Study Of The Catalytic Mechanism Of Myo-Inositol Monophosphatase: Essential Role Of Water In Enzyme Catalysis In The Two-Metal Mechanism. J. Phys. Chem. B 2013, 117 (3), 833−842. (30) Hirao, H. The Effects Of Protein Environment And Dispersion On The Formation Of Ferric-Superoxide Species In Myo-Inositol Oxygenase (Miox): A Combined Oniom(Dft:Mm) And Energy Decomposition Analysis. J. Phys. Chem. B 2011, 115 (38), 11278− 11285. (31) Zhong, L.; Matthews, J. F.; Hansen, P. I.; Crowley, M. F.; Cleary, J. M.; Walker, R. C.; Nimlos, M. R.; Brooks, C. L., III; Adney, W. S.; Himmel, M. E.; et al. Computational Simulations Of The Trichoderma Reesei Cellobiohydrolase I Acting On Microcrystalline

resources and time. This work was supported by 100 Talent Project, the Knowledge Innovation Program of the CAS (Grant No. KSCX2-EW-J-10), National Nature Science Foundation of China (Grant no. 21173247, 31270785 and 21203227), and the Foundation for Outstanding Young Scientist in Shandong Province (No. BS2010NJ020 and JQ201104), and by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.

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