A Lysine Mutation of the Claw-Arm-Like Loop Accelerates Catalysis by

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A Lysine Mutation of the Claw-Arm-Like Loop Accelerates Catalysis by Cellobiohydrolases Zhiyou Zong, Qiyu Li, Zhangyong Hong, Haohao Fu, Wensheng Cai, Christophe Chipot, Huifeng Jiang, Dongyuan Zhang, Shulin Chen, and Xueguang Shao J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 21 Aug 2019 Downloaded from pubs.acs.org on August 21, 2019

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Journal of the American Chemical Society

A Lysine Mutation of the Claw-Arm-Like Loop Accelerates Catalysis by Cellobiohydrolases Zhiyou Zong,a Qiyu Li,a Zhangyong Hong,a Haohao Fu,a Wensheng Cai,*,a Christophe Chipot,b,c Huifeng Jiang,d Dongyuan Zhang,d Shulin Chen,d and Xueguang Shao*,a

a

State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular

Recognition, Research Center for Analytical Sciences, College of Chemistry, Nankai University, Tianjin 300071, China b

Laboratoire International Associé CNRS and University of Illinois at Urbana−Champaign, LPCT, UMR

7019 Université de Lorraine CNRS, Vandœuvre-lès-Nancy F-54500, France c

Department of Physics, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States

d

Key Laboratory of Systems Microbial Biotechnology, Tianjin Key Laboratory for Industrial Biological

Systems and Bioprocessing Engineering, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China

*Corresponding E-mail: [email protected], [email protected]

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ABSTRACT: Searching for viable strategies to accelerate the catalytic cycle of glycoside hydrolase family 7 (GH7) cellobiohydrolase I (CBHI) − the workhorse cellulose-degrading enzymes, we have performed total 12-μs molecular dynamics simulations on GH7 CBHI, which brought to light a new mechanism for cellobiose expulsion, coined “claw-arm” action. The loop flanking the product binding site plays the role of a flexible “arm” extending towards cellobiose, and residue Thr389 of this loop acts as a “claw” that captures cellobiose. Five mutations of residue Thr389 were considered to enhance the loop-cellobiose interaction. The lysine mutant was found to accelerate cellobiose expulsion significantly, and facilitate polysaccharide-chain translocation. Lysine mutation of Thr393 in Talaromyces emersonii CBHI (TeCel7A) performed similarly. Lysine approaches the catalytic area and stabilizes the Michaelis complex, potentially affecting glycosylation, the rate-limiting step of the catalytic cycle. QM/MM calculations indicate that lysine replacement diminishes the barrier against proton transfer, the crucial step of glycosylation, by 2.3 kcal/mol. Experimental validation was performed using the full-length wild-type (WT) of TeCel7A and its mutants, recombinantly expressed in Pichia pastoris, to degrade the substrates. Compared with the WT, the lysine mutant revealed an associated higher enzymatic reaction rate. Furthermore, cellobiose yield was also increased by lysine mutation, indicating that dissociation of the enzyme from cellulose was accelerated, which largely stems from the enhanced flexibility of the “arm”. The present work is envisioned to help design strategies for improving enzymatic activity, while decreasing enzyme cost.

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INTRODUCTION Much attention has been given in recent years to potential applications of lignocellulosic biomass, which can be converted into biofuels and biochemicals, central to the concept of sustainable and renewable energy.1,2 Bioconversion of lignocellulosic biomass in industry remains, however, costly, and enzymatic degradation of cellulose is one of the major cost factors in this process. 3 Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs), catalyzing cellulose depolymerization, are the workhorse cellulose-degrading enzymes, notably cellobiohydrolase I (CBHI or Cel7A).4 Accelerating the catalytic cycle of GH7 CBHI and enhancing their ability to degrade polysaccharides are, therefore, of paramount importance to decrease the enzyme cost. In GH7 CBHI, the polysaccharide chain is degraded in a three-step process after it enters the catalytic tunnel, reaches the designated location, and forms a Michaelis complex (the protein with the catalytically activated polysaccharide chain). Initially, the glycosidic bond between −1 and +1 sugar ring is catalyzed by enzymatic catalytic reaction, i.e., glycosylation and deglycosylation, and the product, i.e., cellobiose, is produced and then expelled from the catalytic tunnel. Finally, the polysaccharide chain moves forward, bringing two glucosyl units (-2 and -1) to occupy the product binding sites (+1/+2), subsequently twists and becomes catalytically activated, known as “processive motion” and “catalytic activation”, respectively.5 The catalytic cycle repeats itself continuously until the enzyme disassociates from the substrate. For accelerating cellobiose expulsion in GH7 CBHI, Bu et al. calculated the absolute binding free-energy of cellobiose to the catalytic tunnel of Trichoderma reesei Cel7A (TrCel7A), employing molecular dynamics (MD) simulations to quantify the inhibitory effect.6 Five mutants were generated computationally, with the aim of decreasing the binding affinity of cellobiose towards the catalytic tunnel, and, hence, accelerating product expulsion. Silveira and Skaf investigated how these 3



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mutations affect the structural and dynamical integrity of the catalytic tunnel. 7 They found that mutation of charged residues into alanine causes severe disruptions to structural integrity, and may weaken the catalytic efficiency of the enzyme. The proposed mutants were generated experimentally by Atreya et al., and their effects on the hydrolytic activity of microcrystalline cellulose (Avicel PH-101) were probed.8 All the mutants suggested by MD simulations ultimately decreased the overall enzymatic activity. The strategy of weakening the interaction of the enzyme with cellobiose, thus, appears unsatisfactory, and ought to be rethought. The overarching goal of the present work is to propose a new strategy for accelerating cellobiose expulsion. To achieve this goal, a detailed understanding of the expulsion process constitutes a prerequisite. We will focus on the loop near the product binding site (Figure 1). How this loop regulates product expulsion remains to be addressed. The present unbiased microsecond-timescale MD simulations of two enzyme-cellobiose assemblies, namely TrCel7A and Talaromyces emersonii Cel7A (TeCel7A), reveal a “claw-arm” action responsible for cellobiose expulsion from the catalytic tunnel. Based on this observation, we propose a mutation strategy aimed at enhancing the loop-cellobiose interaction, in stark contrast with the aforementioned option of decreasing the interaction of cellobiose with the catalytic tunnel. Our next task is to investigate whether the mutated “claw” is able to enter the catalytic tunnel and furnish an additional driving force to enhance the processive motion of the polysaccharide chain. In the GH7 CBHI catalytic cycle, the key step is the enzymatic reaction.9-11 Knott et al. have reported that the free-energy barriers against glycosylation and deglycosylation are 15.5 kcal/mol and 11.6 kcal/mol, respectively.10 Glycosylation, the barrier of which is much higher than that of deglycosylation and polysaccharide chain threading (approximately 4 kcal/mol),5 is recognized as the rate-limiting step in the three-step catalytic cycle of GH7 CBHI. Furthermore, Yan et al. have 4


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calculated the free-energy barriers for proton transfer from the donor residue Glu217 carboxyl group to O4 of the +1 glucosyl ring, and for the nucleophilic attack of Glu212 on C1 in the −1 glucosyl ring for the reaction, indicating that the former, is the crucial step for glycosylation.11 The effect of the proposed mutation strategy on proton transfer was, therefore, investigated via computer simulations, and the associated enzymatic reaction rate was measured in experiments. We also examined the actual hydrolytic power of the mutant on Avicel PH-101 in the absence of synergistic enzymes, and analyzed the possible reasons for the increased cellobiose yield.

RESULTS AND DISCUSSION Spontaneous Process of Cellobiose Expulsion. TrCel7A was used in the present study to decipher the mechanism of cellobiose expulsion (Figure 1). The cellobiose moiety was found to be expelled from the catalytic tunnel around 700 ns (Figure 2a). To rule out the possibility of a random event, two independent MD simulations starting from the protein structure at 600 ns were performed over an additional 200 ns. In each simulation, cellobiose expulsion was observed (see Figure S1 in the Supporting Information).

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Figure 1. Initial structure of TrCel7A (PDB code 4C4D) with a bound polysaccharide chain and a cellobiose unit in the catalytic tunnel. Two leading glucosyl rings cleaved from the polysaccharide chain, forming a cellobiose unit, are numbered +2 and +1, and the remaining rings of the chain are numbered from −1 to −6. Carbon atoms of the polysaccharide chain and the cellobiose are colored in cyan and green, respectively. The catalytic residues (Glu212, Asp214 and Glu217) are highlighted in blue. The loop (T383-A392) near the product binding site, namely A4 loop12 is highlighted in red. The other substrate-enclosing loops A1 (Q98-K102), A2 (T399-S411), A3 (D369-N373), B1 (Y51-W56), B2 (P194-T201), B3 (G244-G253) and B4 (G339-S342) are colored in orange. Thr389 and Arg394 are colored in yellow and magenta, respectively. For clarity, the counterions and water molecules are not shown, here and in the forthcoming figures.

As depicted in Figure 2b, the A4 loop starts to interact with cellobiose after 660 ns, while the interaction of the product and the entire protein diminishes abruptly. This phenomenon suggests that cellobiose has a distinct propensity to release itself from the hindrance of the residues in the catalytic tunnel, leading eventually to its expulsion, by virtue of the attractive interaction with the A4 loop which constitutes the primary driving force for product expulsion. As shown in Figure 2c, it is apparent that Arg394, which is commonly accepted as an important residue that hinders product expulsion (see Figure S2 in the Supporting Information), interacts strongly with cellobiose. However, after about 670 ns such a restraint decreases, while the attraction of Thr389 for cellobiose increases markedly, indicating that this residue starts to compete with Arg394 for the product. Finally, the cellobiose moiety is no longer attracted by Arg394. Thr389 in the middle of the A4 loop may, therefore, be considered as a key residue in cellobiose expulsion.

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Figure 2. Spontaneous cellobiose expulsion from the catalytic tunnel. (a) Time evolution of the distance between the center-of-mass (COM) of cellobiose and the −1 glucosyl ring over the 600-700 ns period. A distance greater than 16 Å indicates expulsion. (b) Interaction energies of the A4 loop and of the entire protein with cellobiose. (c) Interaction energies of residues 385 to 394 with cellobiose. (d) Root mean square fluctuation (RMSF) values of the residues of TrCel7A with and without the cellobiose in the product binding site obtained from 600-ns trajectories. (e-h) Snapshots of the cellobiose expulsion. The analyses of the 4C4D assembly are based on the 1.0-μs MD trajectory, and those of the cellobiose-free 4C4D assembly, on the 600-ns MD trajectory.

As illustrated in Figure 2d, compared to the cellobiose-free assembly, the A4 loop appears to be more flexible when cellobiose is located at the product binding site. Furthermore, residues 386 to 390 possess the highest flexibility. It is, therefore, reasonable to believe that high mobility and ability to form electrostatic interactions (see Figure S3 in the Supporting Information) confer to Thr389 a prominent role. Based on the aforementioned analyses, a “claw-arm” action of cellobiose expulsion is proposed. The A4 loop plays the role of a flexible “arm” to reach the product binding site and interact with cellobiose. Residue Thr389 in this loop acts as a “claw” that captures cellobiose, and facilitates the transfer of the product into the aqueous environment, completing the expulsion. Four representative snapshots of cellobiose expulsion generated from the MD trajectory are gathered in 7



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Figure 2e-h (see movie M1 in the Supporting Information). Site-Directed Mutations as a Route to Accelerate Cellobiose Expulsion. It is apparent from the above analysis that the approach of Thr389 towards cellobiose, which competes with that of Arg394, is much slower than the release of the product from the A4 loop. Thus, accelerating the former process constitutes a critical step to speed up product expulsion. Although mutating Arg394 into alanine, with the aim of weakening its interaction with cellobiose, should benefit the competition of Thr389 against Arg394, the processive motion of the polysaccharide chain may be limited.8 Therefore, we put forth a distinct strategy for enhancing the binding of Thr389 with cellobiose and further with the polysaccharide chain through mutation of that particular residue. The criteria for the mutation are that the replacement residue should possess (i) the ability to form strong electrostatic interactions with cellobiose and the polysaccharide chain, and (ii) a long side chain, so that it can approach cellobiose easily and enter deeper the catalytic tunnel to pull the polysaccharide chain. Five mutations at position 389 were proposed, including two positively charged residues, i.e., T389R and T389K, one neutral one, i.e., T389Q, and two negatively charged ones, i.e., T389D and T389E.

Figure 3. Effect of the mutations on cellobiose expulsion. (a) Distance separating the COMs of the cellobiose moiety from that of the −1 glucosyl ring before and after mutations. (b) Interaction energies of the residue at 8


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position 389 in TrCel7A WT and mutants with cellobiose. (c) Interaction energies of T389K and Arg394 with cellobiose. (d) Comparison of the RMSF values of the residues between the WT and the five mutants based on 150-ns trajectories before cellobiose expulsion. (e-h) Snapshots from the MD trajectories depicting the interaction of T389K and Arg394 with cellobiose. The analyses are based on the 300-ns MD trajectories of the T389R, T389K, T389D, T389E and T389Q assemblies.

It can be seen from Figure 3a that only with T389K, is cellobiose expelled from the catalytic tunnel within 200 ns. Expulsion of cellobiose is also achieved in two of the four independent 300-ns simulations of T389K (see Figure S4 in the Supporting Information). This observation can be rationalized by a more favorable interaction of the product with T389K, compared with the other mutants, as shown in Figure 3b. Moreover, this favorable interaction finally led the cellobiose moiety to leave the tunnel within 170 ns, after competing with Arg394, as illustrated in Figure 3c. Figure 3d indicates that the flexibility of the A4 loop after mutation into T389K increases significantly. This enhanced flexibility, which has no bearing on cellobiose unbinding, will not only help lysine to approach the product, but also the loop to carry cellobiose out of the catalytic tunnel. As shown in Figure 3e-h, after leaving Arg394 and interacting with the T389K, cellobiose quickly moves away from the A4 loop into the aqueous environment (see movie M2 in the supporting Information). In our 300-ns simulations, why did T389K accelerate cellobiose expulsion from the catalytic tunnel, when T389R, T389Q, T389D and T389E did not? The electrostatic potentials mapped on the surfaces of the product binding site and the five mutated residues (see Figure S5 in the Supporting Information) indicate that the attraction of the positively charged T389K by the electronegative environment helps this residue approach the product binding site, and form strong hydrogen-bonding 9



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interaction with cellobiose (see Figure S6 in the Supporting Information). Yet, another positively charged residue, namely T389R, which is very similar to T389K, does not form as strong an interaction with cellobiose as T389K (Figure 3b). A reasonable explanation is that the two NH2 groups of the guanidinium moiety sterically hinder entry into the catalytic tunnel, weakening the interaction with the substrate. Compared with the charged residues, the electrostatic interaction of the neutral T389Q and cellobiose is similar to that of the WT. The electrostatic repulsion between T389D or T389E and the product binding site decreases the probability of hydrogen bonding with cellobiose. Investigation of the “Claw-Arm” Action in GH7 CBHI. The structure-based sequence alignment of eleven GH7 CBHs (including eight GH7 CBHI) performed by Hobdey et al.13 indicates that at position 389 in the A4 loop of TrCel7A, threonine also exists in GH7 CBHI from four alternate

organisms

(T.

emersonii,

Trichoderma

harzianum,

Aspergillus

fumigatus

and

Heterobasidion irregulare), as well as aspartic acid in Dictyostelium discoideum CBHI, glutamic acid in Dictyostelium purpureum CBHI, and lysine in Humicola grisea var. thermoidea CBHI. Our in-silico mutations of residue 389 demonstrate that, compared with threonine, only lysine markedly enhances the loop-cellobiose interaction, while aspartic acid and glutamic acid do not. Does the threonine-to-lysine mutation at this key position in the A4 loop of other GH7 CBHI possess the same capability to accelerate product expulsion out of the catalytic tunnel? To answer this question, we investigated TeCel7A, which not only has a 66% sequence identity with TrCel7A, but also corresponds to a starkly similar three-dimensional structure. The sequence and structure alignments between the two proteins are provided in Figure S7 and Figure S8 in the Supporting Information. To address whether or not the A4 loop in TeCel7A possesses the same “claw-arm” function as in TrCel7A, MD simulations of spontaneous cellobiose expulsion were performed for the WT 10


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TeCel7A and its T393K mutant. Analysis of the MD trajectories reveals that mutating T393 into lysine also accelerates product expulsion from the catalytic tunnel (see Figure S9a in the Supporting Information). This spontaneous expulsion was observed in one of the three independent 300-ns simulations. Flexibility of the A4 loop (see Figure S9b in the Supporting Information) is enhanced as well, although not as glaringly as in TrCel7A by T389K (see Figure 3d). These results, together with those for TrCel7A, underscore the potential effect of the A4 loop on cellobiose expulsion in the GH7 CBHI family, and that the threonine-to-lysine mutation possibly represents a strategy evolved in this family of enzymes to speed up product expulsion out of the catalytic tunnel. “Claw-Arm”-Mediated Cellobiose Expulsion. The two-dimensional free-energy landscapes underlying cellobiose expulsion from the catalytic tunnel coupled with residue 389 extending towards the product are depicted in Figure 4a and b for the WT and the lysine mutant. From the determined least free-energy pathways for cellobiose expulsion (Figure 4c), we found that for the WT, (i) threonine has a propensity to approach the product binding site and capture cellobiose (black dotted line in Figure 4a), (ii) the barrier against product expulsion is about 1.9 kcal/mol. The pathway for T389K depicted in Figure 4b is significantly different. Lysine can approach the product binding site and capture cellobiose with a favorable transfer free energy. Furthermore, throughout the entire process, lysine interacts strongly with cellobiose. The distinct pathway and the strong interaction with the product resulting from lysine replacement lead to a reduced free-energy barrier by 0.8 kcal/mol against expulsion, compared with the WT (Figure 4c). Although the reduction of the free-energy barrier is minor, it is still statistically significant, and consistent with the decreased cellobiose expulsion time (about 500 ns) observed in our MD simulations.

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Figure 4. Free-energy landscapes characterizing the cellobiose expulsion averaged over two independent runs, in the (a) WT and (b) T389K assemblies. The black lines highlight the least free-energy pathways for cellobiose expulsion. S0 corresponds to the initial position that cellobiose locates at the product binding site and residue 389 is far away from this site, and S1, the final stable position that cellobiose is expelled from the catalytic tunnel. In Figure 4(a), S0 (d1, ξ1) = (15.0 Å, 6.0 Å) and S1 (d1, ξ1) = (14.0 Å, 20.0 Å). In Figure 4(b), S0 (d1, ξ1) = (18.0 Å, 6.0 Å) and S1 (d1, ξ1) = (14.0 Å, 20.0 Å). (c) Free-energy profiles for cellobiose expulsion as a function of the position (s) along the least free-energy pathways. s = 0.0 and s = 1.0 represent the locations S0 and S1, respectively. Two primary coarse variables (d1, ξ1) are utilized to explore the putative transition pathways. The error bars correspond to the standard deviation of the mean inferred from two independent 500-ns runs for each molecular assembly. (d, e) d1 denotes the distance between the COMs of the cellobiose moiety and residue 389. d1 ˂ 11 Å in Figure 4a, and ˂ 14 Å in Figure 4b indicate that Thr389 and T389K capture the cellobiose, respectively. (f) ξ1 denotes the projection onto the longitudinal axis of the catalytic tunnel of the distance separating the COM of the cellobiose moiety from that of the −1 glucosyl ring. ξ1 > 16 Å indicates cellobiose expulsion from the catalytic tunnel.

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Bu et al. have reported that the absolute binding free energy of cellobiose to the product binding site is 11.2 kcal/mol, using alchemical free energy perturbation calculations,6 which we believe to be grossly overestimated.5 In our calculations, owing to the “claw-arm” action, the free-energy barrier against cellobiose expulsion is very low, and the overall process is thermodynamically favorable (Figure 4c), which is also reflected in our MD simulation of spontaneous expulsion. We conjecture that except for the different initial structures,10 the lack of synergy between the A4 loop and cellobiose expulsion constitutes the main reason for this discrepancy. Processive Motion of the Polysaccharide Chain. For the sake of investigating the effect of lysine mutation on the processive motion of the polysaccharide chain after cellobiose expulsion, an additional 700-ns simulation of T389K was performed. As shown in Figure 5a and b, T389K is found to enter deeply the catalytic tunnel, and form strong electrostatic interactions with the −1 glucosyl ring of the polysaccharide chain (see Figure S10 and movie M3 in the Supporting Information). Lysine mutation on Thr393 in TeCel7A performs similarly (see Figure S11 in the Supporting Information). This interaction promotes pulling of the polysaccharide chain, which moves about one glucosyl ring within 1.0 μs, whereas, that chain in the WT hardly moves in the TrCel7A (see Figure S12 in the Supporting Information). With the movement of T389K, the entire loop moves closer to the product binding site after 200 ns, especially around 500 ns (Figure 5c), conducive to the formation of contacts between Arg394 and the −1 glucosyl ring (Figure 5d). The attraction of both Arg394 and T389K to the −1 glucosyl ring provides the thermodynamic driving force for the continuous processive motion of the polysaccharide chain. After the -1 and -2 rings occupy the product binding site, the polysaccharide chain is catalytically activated, preparing for the next step, i.e., glycosylation. The effect of lysine mutation on the catalytic activation falls beyond the scope of the present work. 13



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Figure 5. Effect of T389K mutation on processive motion of the polysaccharide chain. (a) Snapshot of T389K interaction with −1 glucosyl ring of the polysaccharide chain. (b) Interaction between T389K and the −1 glucosyl ring of the polysaccharide chain. Before (black line) and after (blue line) mutation, distance separating the C1 atom of the −1 glucosyl ring from the COMs of (c) the A4 loop and (d) the two NH2 groups of Arg394. The analyses are based on the 1.0-μs MD trajectories of WT and T389K, separately.

Effect of Lysine Mutation on Proton Transfer during Glycosylation. The rate-limiting step in the three-step catalytic cycle of CH7 CBHI is glycosylation,5, 9-11 wherein, proton transfer has been identified as the rate-limiting step of the reaction.11 We, therefore, employ QM/MM simulations combined with free-energy calculations to investigate whether lysine mutation can affect proton transfer. First, a 300-ns free-energy calculation was performed for the TrCel7A structure (PDB code 8CEL) with T389K mutation, wherein the polysaccharide chain was catalytically activated, to determine the PMF describing Lys389 entering the catalytic tunnel (see Figure S13 in the Supporting Information). Lys389 was found to approach the catalytic region at a marginal energetic cost, and stabilize the Michaelis complex. The structure corresponding to the minima of the PMF is used as the initial model in the following QM/MM calculations for T389K. Detail of these simulations is provided in the section of Methods and the Supporting Information.

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Figure 6. The selected atoms in (a) the WT and (b) T389K for QM calculations. Some hydrogen atoms are not shown for clarity. The polysaccharide chain in the structure of 8CEL becomes catalytically activated (−1 sugar ring possesses a “half-chair” conformation). Carbon atoms of substrate and residues are shown in green and in yellow, respectively. C1 in −1 glucosyl ring and O4 in +1 glucosyl ring are highlighted in ball-and-stick, and the bond between them will be broken. (c) Comparison between the free-energy profiles characterizing proton transfer reactions for the WT and T389K generated from QM/MM simulations. The error bars correspond to the standard error inferred from three independent 50 ps runs for each molecular assembly. d3 denotes the distance between the H atom of Glu217 and O4 of the +1 glucosyl ring.

The glycosylation mechanism of TrCel7A has been studied by several groups.9-11 Selection of collective variables to form an optimal transition coordinate characterizing glycosylation has been investigated in detail by Knott et al.10 In the work of Yan et al., the Glu217 H − O4 in +1 ring distance was employed to describe proton transfer.11 Here, the same transition coordinate (d3 in Figure 6) was used, along which the PMFs describing proton transfer were determined. In our simulations, proton transfer and cleavage of the glycosidic linkage were found to be concerted (see movie M4 in the Supporting Information). QM/MM calculations for the WT (Figure 6a) and T389K (Figure 6b) indicate that the barrier against proton transfer resulting from lysine mutation is lowered by 2.3 kcal/mol, relative to the WT (Figure 6c). The positive charge borne by the lysine residue, 15



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which balances the charge of the QM region to zero, thus modulating the electrostatic potential in the catalytic area, may be responsible for the smaller free-energy barrier. However, it should be noted that the high flexibility of the A4 loop precludes an extremely steady anchoring of the lysine residue in the catalytic tunnel, which is also mirrored in the PMF of Figure S13. The effect of lysine mutation on glycosylation, therefore, depends on the frequency at which lysine enters and leaves the tunnel. Still, lysine mutation constitutes a promising strategy to enhance enzymatic hydrolytic efficiency. Effect of Lysine Mutation on Enzymatic Reaction Rate. To investigate the effect of the lysine-mutation strategy elaborated from the above simulations on the three-step catalytic cycle of GH7 CBHI, the full-length WT TeCel7A (see Figure S14 in the Supporting Information) and mutants were synthesized, expressed in Pichia pastoris and purified (see Figure S15 in the Supporting Information). Enzymatic kinetics experiments with these enzymes were carried out by hydrolysis of soluble substrate of p-nitrophenol-cellobioside (pNPC) to measure the reaction rate. T393K has a higher reaction rate than the WT (see Table 1), which can be rationalized by the accelerated proton transfer resulting from lysine mutation, as evidenced by our QM/MM calculations.

Table 1. Enzymatic Kinetics for the WT and T393K Km (mM)

kcat (min-1)

kcat/Km (min-1 mM-1)

WT

1.03

4.13

3.99

T393K

1.04

5.1

4.87

Effect of Lysine Mutation on Degradation of Avicel PH-101. The above three-step catalytic cycle of GH7 CBHI will repeat itself 10-60 times as the insoluble substrate, i.e., cellulose, is degraded.5, 14 Then, the enzyme will undergo two non-catalytic processes, i.e., (i) the dissociation of 16


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the enzyme from the substrate when encountering an obstacle, and (ii) the association of the enzyme to the substrate again in anticipation of the next catalysis.15, 16 In the absence of synergistic enzymes (e.g., endoglucanases, and non-reducing end specific CBHs), the rate of the enzyme leaving substrate after catalysis, i.e., the dissociation rate (koff), is known to be the rate-limiting step for GH7 CBHI degrading cellulose.12, 14, 17-22 Taylor et al. have reported that the flexibility of the substrate-enclosing loops (see Figure 1) has a prominent effect on koff, and high flexibility mediates enzyme dissociation from cellulose.3 As shown in Figure 3d and Figure S9b, the flexibility of A4 loop both in TrCel7A and TeCel7A is enhanced significantly owing to lysine mutation. Further analysis demonstrates that flexibility of loops A2 and A3 is increased as well, owing to the influence of the highly flexible A4 loop (Figure 7a). Conversely, the flexibility of the substrate-enclosing loops in other mutants is not enhanced as significantly as that in the lysine mutant (see Figure S16 in the Supporting Information). The actual performance of the WT TeCel7A and the relative mutants on insoluble substrate was investigated via degrading Avicel PH-101. As reported in Figure 7b, the lysine mutant shows superior performance compared to the other mutants, resulting in a 270 mg/L cellobiose release, while the WT releases 210 mg/L cellobiose. This result indicates that the increase in flexibility of the substrate-enclosing loops contributes to accelerate the dissociation of the enzyme from the substrate, and thus, enhance the ability of GH7 CBHI to degrade cellulose.

Figure 7. (a) Comparison of the RMSF values of the residues between the TrCel7A WT and the lysine mutant. The 17



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loops A2 A3 and A4 are highlighted in red. (b) Cellobiose released from Avicel PH-101 by mutants (T393R, T393K, T393D, T393E, T393Q and T393A) and WT TeCel7A after 60 hours at 60 °C. T393A was constructed to assess the influence of Thr393 on the flexibility of the A4 loop through inactivating it. Experiments were performed in triplicate and error bars represent the standard error of the mean.

CONCLUSIONS In the present work, a new mechanism for cellobiose expulsion, which we coin “claw-arm” action, is proposed, expanding and deepening our knowledge of the role played by the A4 loop on the CH7 CBHI catalytic cycle. In stark contrast with previous strategies for accelerating product expulsion, we put forth a mutation strategy aimed at enhancing the A4 loop-cellobiose interaction. Lysine mutation on this “claw” site accelerates cellobiose expulsion, facilitates polysaccharide-chain translocation, and decreases the barrier against proton transfer, resulting in a higher enzymatic reaction rate. The A4 loop possesses high flexibility, owing to the lysine mutation, which increases cellobiose yield. In conclusion, coordination of the strong lysine-“claw” and highly flexible “arm” contributes to enhance the enzymatic efficiency and to decrease the enzyme cost. In view of the industrial importance of the family of enzymes examined here, performance of the lysine mutant versus the WT in an industrial setting might be a matter of concern. Yet, bioconversion of lignocellulosic biomass in industry is a complex process. Modification of the enzyme-cocktail composition or of the substrate types necessarily results in a change in the rate-limiting step of the enzymatic processive cycle. These questions of industrial relevance fall, however, beyond the scope of this work, and deserve a separate study in their own right. Still, the present work, at the confluence of theory and experiment, is anticipated to help design more efficient enzymes for cellulosic bioconversion. 18


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METHODS Simulation Design. The crystal structure of TrCel7A (PDB code 4C4D), including a polysaccharide chain and a cellobiose unit (the product cleaved from the polymer chain) in its catalytic tunnel (see Figure 1), was solvated in an equilibrated box of water, and the overall charge neutrality was achieved by adding Na+ ions to the solution. The total molecular assembly contains about 56,000 atoms. To investigate the entire cellobiose expulsion process and the ensuing motion of the polysaccharide chain, a 1.0-μs MD simulation of this assembly was carried out. Moreover, to explore the effect of cellubiose on the loop, a cellobiose-free model was constructed by removing the cellubiose molecule from the initial structure of 4C4D, followed by a 600-ns MD simulation. It should be noted that in 4C4D Glu217 has been mutated into Gln217. Our additional simulation, changing Gln217 back into Glu217, demonstrates that this mutation hardly affects the action observation of cellobiose expulsion in the MD simulations (see Figure S17 in the Supporting Information). In order to increase the attractive interaction of the loop with cellobiose, five mutations of key residue Thr389 were designed. A 300-ns MD simulation was conducted and repeated at least two times for each mutant as a way to discriminate the most promising one to accelerate cellobiose expulsion. The timescale of the simulation for the best mutant was expanded to 1.0 µs to probe the subsequent processive motion of the polysaccharide chain. The crystal structure of TeCel7A (PDB code 3PFX) with a cellobiose unit and a polysaccharide chain, which was extracted from TrCel7A and inserted at the corresponding position in the catalytic tunnel of TeCel7A, was used as the initial structure for cellobiose-expulsion simulations. Then, the cellobiose molecule was removed for investigating the processive motion of polysaccharide chain. The WT TeCel7A and its T393K mutant 19



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in explicit solvent were built using the same method as for TrCel7A. MD simulations for cellobiose expulsion and polysaccharide-chain translocation of the two assemblies were repeated at least twice and once, respectively. All the brute-force MD simulations reported in this study were carried out using the scalable program NAMD 2.1223 with the CHARMM36 force field,24-26 and the TIP3P water model.27 Visualization and analysis of the MD trajectories were performed with the VMD program. 28 Each molecular system before production simulations underwent a standard operation procedure.10 A total of 14 molecular assemblies were simulated over an aggregate time of approximately 12 μs (details are provided in Table S1 of the Supporting Information). Free-Energy calculations. The two-dimensional free-energy landscapes characterizing the cellobiose expulsion coupled with residue 389 extending towards the product binding site were generated utilizing the well-tempered meta-eABF (WTM-eABF) method.29, 30 Instantaneous values of the force were stored in bind, with 0.1 Å × 0.1 Å wide. The algorithm of Minimum Energy Path Surface Analysis31 is used to determine the least free-energy pathways of the associated motions from the two-dimensional free-energy maps. In addition, the potential of mean force (PMF) describing the T389K in TrCel7A (PDB code 8CEL) entering the catalytic tunnel was determined to probe whether or not lysine can approach the catalytic area when the polysaccharide chain becomes catalytically activated. The total calculative time amounted to 2.3 μs. Detail of each molecular assembly is provided in Table S2 of the Supporting Information. QM/MM Free-Energy Calculations. The PMFs of proton transfer during glycosylation were determined for the WT (PDB code 8CEL) and T389K in aqueous solution, by quantum mechanics/molecular mechanics (QM/MM) simulations. The QM region of the WT assembly, which has an overall charge of -1, includes Glu212, Asp214, Glu217, as well as the −1 and +1 glucosyl 20


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units. The QM area of T389K, which has an overall charge of 0, contains the QM atoms of the WT and a lysine residue. The initial structure of T389K for the QM/MM calculation is generated from the stable state of the complex after lysine enters the catalytic area, which is determined by the above PMF calculation. The WTM-eABF method was employed in all the PMF calculations. The transition pathway, extending from 2.0 to 1.0 Å was broken down into five windows for increasing the efficiency of the calculations. In the QM/MM calculations, electrical embedding was employed to treat Coulombic interactions at the QM/MM interface.32,

33

Considering the calculation accuracy and cost，the

semiempirical method, PM7,34 was used in the QM calculations. MOPAC35 and NAMD were utilized as the QM and MD engines, respectively. Instantaneous values of the force were accrued in bins 0.01 Å wide. Three independent runs of the QM/MM simulation for each molecular assembly were carried out with a 0.02 fs time step. The total calculative time amounted to 300 ps. Detail of each molecular assembly is exhibited in Table S3 of the Supporting Information. Construction of TeCel7A. TeCel7A is not only very similar in both sequence and structure to TrCel7A (see Figure S7 and Figure S8 in the Supporting Information), but also easier to express than the latter. The former protein was, therefore, used in this work to experimentally examine the effect of the mutants on the substrates. The mature amino acid sequence of TeCel7A contains a catalytic domain (CD), a carbohydrate binding module (CBM) and a short, flexible linker, which connects CBM and CD, as depicted in Figure S14 of the Supporting Information. The WT and six mutants of T393R, T393K, T393D, T393E, T393Q and additional T393A were generated by total gene synthesis and connected to yeast expression vector pPICZαA (Invitrogen, Carlsbad, CA). The codons were optimized, and His-tag was added to the C-terminal for purification. All the synthetic experiments were carried out at the Beijing Genomics Institute. 21



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Expression of Enzymes. The recombination genes were linearized by Sac I (Thermo Fisher Scientific, Runcorn, Cheshire, UK), and transformed into the protein production host organism X-33 (Invitrogen, Carlsbad, CA) by electroporation.36 Transformants were spread on YPDS plates (1% yeast extract, 2% peptone, 2% glucose, 1 M sorbitol, 2% agar) containing 100 μg/ml zeocin (Thermo Fisher Scientific, Runcorn, Cheshire, UK) and incubated for three days at 30 °C. The single colonies of the WT and mutants from the plates were inoculated into buffered complex medium containing glycerol (BMGY) and grew overnight at 30 °C with shaking at 240 rpm until the cell turbidity OD reached to 2-6. The cells were then pelleted via centrifuging at 4,000 g for 15 minutes and resuspended into buffered minimal medium containing methanol (BMM). The cultures were induced by supplementing 0.5 % methanol every 12 hours and grew for three days at 30 °C, 240 rpm. Purification of Enzymes. Following protein expression, cultures were centrifuged at 4,000 g for 30 minutes to clarify the supernatants containing the enzymes. The supernatant for each variant was adjusted to pH 7.0 by adding 0.1 M NaOH solution, and then filtered by 0.45 μm polypropylene membranes (Seahorse Bioscience, North Bellerica, MA) to remove residual cells. The enzymes were purified from the cultures by a cOmplete His-Tag Purification Resin (Roche, Mannheim, Germany). The enzyme was adsorbed onto nickel powder (2 mL) by adding the supernatant to the column continuously and the impurities were removed by phosphate buffer saline (PBS), pH 7.0, and PBS containing 5 mM imidazole successively. Then 2 ml PBS containing 300 mM imidazole was utilized to elute the enzymes. Samples were concentrated and buffer exchanged into 50 mM citrate buffer, pH 5.0 using Vivaspin 30K MWCO PES centrifugal concentrators (Sartorius, Concord, CA). The purity of the enzymes was analyzed by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) (see Figure S15 in the Supporting Information). The concentration of purified enzymes 22


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was determined using a Nanodrop-2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Enzymatic kinetics. The enzymatic kinetics was measured with pNPC (Sigma, St. Louis, MO) as the substrate. The concentration of pNPC ranges from 0.1 to 1 mg/mL in 100 μL reaction systems, 50 mM citrate buffer, pH 5.0, with 1.33 μM purified enzymes of the WT and the mutant, separately. After incubation at 60 °C for 10 minutes, these reactions were terminated by adding 100 μL of 10% Na2CO3. The amount of released pNP from the enzymatic reaction was estimated based on the absorbance at 420 nm. The inactive enzyme boiled for 10 minutes at 100 °C was utilized as the control. Quantification was performed by external calibration with a set of pNP solutions ranges of 0.6–3 μg/mL. Kinetic parameters kcat and Km were estimated by measuring the initial velocities of enzymatic reaction and curve-fitting according to the Michaelis-Menten equation, using GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA). All experiments were performed in triplicate. Degradation of Avicel PH-101. Assays to measure the ability of purified enzymes on cellulose degradation were performed in 1 mL reaction volumes with 10 mg/mL Avicel PH-101 (Sigma, St. Louis, MO) substrate and 1.33 μM purified enzyme in 50 mM citrate buffer, pH 5.0. All experiments were performed in triplicate, and incubated for 60 hours at 60 °C with constant rotational mixing, and finally boiled for 5 minutes at 95 °C to stop the reactions. Assay of the Activity. The samples were centrifuged at 8,000g for 10 minutes, and then filtered using 0.25 μm polypropylene membranes (Seahorse Bioscience, North Bellerica, MA). Ultra Performance Liquid Chromatography (UPLC) system (Waters, Milford, Massachusetts, USA) consisting of refractive index (RI) detector was used to quantify the cellobiose concentrations in the reactions. 20 μL supernatant was injected onto a 250 × 4.6 mm (length × inner diameter) Supersil 23



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NH2-S 5 μm column (Elite, Dalian, China). Compounds were eluted at a flow rate of 1.0 mL using a mobile phase of ultrapure water with 65% acetonitrile. Quantification was performed by external calibration with a set of cellobiose solutions in the range of 100–1000 mg/L.

Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org. Additional information related to the Methods and Results and Discussion sections (PDF) Movies M1−M4 (ZIP)

ACKNOWLEDGMENTS The authors thank Prof. Gregg T. Beckham and associate Prof. Shi H. Yan for their helpful suggestions. This study was supported by the National Natural Science Foundation of China (21773125 and 21775076), the Natural Science Foundation of Tianjin, China (18JCYBJC20500), the Special Program for Applied Research on Super Computation of the NSFC-Guangdong Joint Fund (Second Phase) under Grant U1501501, and “the Fundamental Research Funds for the Central Universities”, Nankai University (63191361). Z. Z. gratefully acknowledges the financial support from China Scholarship Council (201706205014). C. C. is indebted to the Centre National de la Recherche Scientifique for the support of his joint research program (PICS) with the People’s Republic of China.

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A Lysine Mutation of the Claw-Arm-Like Loop Accelerates Catalysis by Cellobiohydrolases

Zhiyou Zong, Qiyu Li, Zhangyong Hong, Haohao Fu, Wensheng Cai, Christophe Chipot, Huifeng Jiang, Dongyuan Zhang, Shulin Chen, and Xueguang Shao

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A Lysine Mutation of the Claw-Arm-Like Loop Accelerates Catalysis by

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