MM and MM MD Simulations on the Pyrimidine-Specific

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QM/MM and MM MD Simulations on the PyrimidineSpecific Nucleoside Hydrolase: A Comprehensive Understanding Toward Enzymatic Hydrolysis of Uridine Fangfang Fan, Nan-Hao Chen, Yong-Heng Wang, Ruibo Wu, and Zexing Cao J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b10524 • Publication Date (Web): 29 Dec 2017 Downloaded from http://pubs.acs.org on December 30, 2017

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The Journal of Physical Chemistry

. QM/MM and MM MD Simulations on the Pyrimidine-Specific Nucleoside Hydrolase: A Comprehensive Understanding toward Enzymatic Hydrolysis of Uridine Fangfang Fan,a Nanhao Chen,c Yongheng Wang,b Ruibo Wub and Zexing Cao*a a.

State Key Laboratory of Physical Chemistry of Solid Surfaces and Fujian Provincial

Key Laboratory of Theoretical and Computational Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 360015, China b.

School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006,

China c.

Department of Chemistry, University of California, Davis, California 95616, United

States

ABSTRACT: The pyrimidine-specific nucleoside hydrolase Yeik (CU-NH) from Escherichia coli cleaves the N-glycosidic bond of uridine and cytidine with a 102~104-fold faster than that of purine nucleoside substrates such as inosine. Such remarkable substrate specificity and the plausible hydrolytic mechanisms of uridine have been explored by using QM/MM and MM MD simulations. The present calculations show that the relatively stronger hydrogen bond interactions between uridine and the active-site residues Gln227 and Tyr231 in CU-NH play an important role in enhancing the substrate binding and thus promoting the N-glycosidic bond cleavage, in comparison with inosine. The estimated energy barrier of 30 kcal/mol for the hydrolysis of inosine is much higher than 22 kcal/mol for uridine. Extensive MM MD simulations on the transportation of substrates to the active site of CU-NH indicate that the uridine binding is exothermic by ~23 kcal/mol, more remarkable than inosine (~12 kcal/mol). All of these arise from noncovalent interactions between the substrate and the active site featured in CU-NH, which account for the substrate specificity. Quite differing from other nucleoside hydrolases, here the enzymatic N-glycosidic bond cleavage of uridine is less influenced by its protonation.

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1. Introduction Nucleoside hydrolases (NHs) are calcium-dependent enzymes that are able to cleave the C-N glycosidic bond of ribonucleosides to generate free ribose and nitrogenous bases (Figure 1),1-5 and this enzymatic hydrolysis is critical to the survival of certain organisms lacking a de novo biosynthetic pathway for purines and pyrimidines. These nucleoside hydrolases, widely existed in nature, have been found not only in different protozoa,5-6 but also in bacteria,7-8 insects,9 yeast,10-12 and nematodes. Whereas these hydrolases have never been observed in mammals,13 they become excellent targets for the antiparasitic drug design. Based on the difference in substrate specificity, the NHs can be classified into four subclasses, including the pyrimidine-specific cytidine-uridine nucleoside hydrolase

(CU-NH),11-12,

14

the

nucleoside hydrolase (IAG-NH),6,

purine-specific

15-17

inosine-adenosine-guanosine

the 6-oxo-purine-specific inosine-guanosine

nucleoside hydrolase (IG-NH),18-19 and the nonspecific inosine-uridine nucleoside hydrolase (IU-NH),5, 20-21 which hydrolyzes both purine and pyrimidine nucleosides. Alternatively, on the basis of the sequence features and the active site residues from the primary structures of the representative NH proteins, the nucleoside hydrolases approximately fall into three homology-based groups. Group I consists of the pyrimidine-specific and nonspecific nucleoside hydrolases (IU-NH and CU-NH). Group II contains the purine-specific IAG-NH, and a yet uncharacterized subfamily belongs to Group III, where a cysteine residue takes the catalytic His239 site of Group I.21 In 2004, the crystal structure of the Escherichia coli YeiK protein in complex with inosine at 1.7 Å resolution provides the first structural information of a pyrimidine-specific NH,22 and after a few years, the X-ray structures of the CU-NH YeiK at the apo state and in complex with the transition-state-like inhibitor have also been determined.23 These structures reveal that the NH family generally has a divalent calcium ion bound at the active site, and the pyrimidine-specific CU-NH YeiK has a similar structure and composition with the IU-NH, differing from the IAG-NHs.24-25 However, the key active-site residues accounting for the substrate specificity and plausible catalytic mechanisms of the CU-NHs are still less known, compared to the IU-NHs.26-34

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In catalysis by NHs, it was assumed that the substrate protonation prior to the transition-state formation plays an important role in activation of the leaving group, but the corresponding detailed mechanisms are still controversial. Our previous MM and QM/MM MD simulations on the enzymatic hydrolysis of the N-glycosidic bond by IAG-NH show that the protonation at the N7 site of inosine can remarkably reduce the free energy barrier for the chemical step, and the rate-determining step for the whole enzymatic catalysis is the ribose release, instead of the N-glycosidic bond cleavage and the base release.35-37 In comparison with such well-characterized NHs, no knowledge of the mechanistic details for the pyrimidine-specific CU-NH is available. Furthermore, what’s the substrate protonation effect on activation of the leaving group in the enzymatic hydrolysis of uridine by CU-NH here? The QM/MM strategy to combine the accuracy of QM methods and the computational efficiency of MM approaches has been proved to be powerful computational tool to deal with the enzymatic reactions.38-43 Herein extensive MM and QM/MM MD simulations on the cleavage of C-N glycosidic bond by CU-NH have been performed, and plausible enzymatic mechanisms and roles of several conserved residues towards the substrate specificity have been explored. Based on QM/MM calculations and MD simulations, the substrate delivery to the active site and the effects of protonation at various sites of substrate on the activation of leaving group have been discussed.

Figure 1. The NH-catalysed hydrolysis of substrates uridine and inosine. 2. Computational details Setup of the Enzyme-Substrate Complex Model The initial enzyme-substrate models were prepared based on the crystal structure 3



of the YeiK complex with an inhibitor at 2.0 Å resolution (PDB ID: 3MKN).23 A tetramer of YeiK indicates that the A/C (or B/D) dimer has more extensive monomer-monomer interactions with an interface surface of 1071 Å2 than the A/B (or C/D) dimer with an interface surface of 891 Å2.14 In order to reduce computational costs, here only the A/C dimer of the YeiK tetramer was used for the computational model (refer to Figure S1 in the Supporting Information), in which the unresolved flexible loops in the subunit were repaired based on the available complete structure of the chain, and the inhibitor was modified into the uridine substrate. The substrates in both chains and the protein were described by the AMBER GAFF force field44 and the AMBER99SB force field,45-46 respectively. The partial atomic charges of substrate were assigned by the restrained electrostatic potential (RESP) charge47 at the HF/6-31G* level with the Gaussian03 package.48 The whole system was solvated into a ∼81 × 77 × 110 Å rectangular box of TIP3P water49 and 24 sodium ions were added at the protein surface to neutralize the model system. The resulting system has a total of about 90000 atoms. The initial coordinates and topology parameters were generated by the tleap tool in AMBER12.50 During our investigation on the substrate specificity, the 2.3 Å crystal structure of the YeiK-inosine complex (PDB: 3B9X)22 was used to prepare various enzyme-substrate systems, and the anti- and syn-conformations of pyrimidine nucleoside (uridine) with different protonation states were also considered. MM MD and RAMD-MD Simulations All models are optimized by the energy minimization at the MM level at first, and then the system was heated up from 0 to 300K gradually under the NVT ensemble for 100 ps. Another 100 ps MD simulation was performed under the NPT ensemble to relax the system density to about 1.0 g/cm3, with the target temperature and pressure of 300K and 1.0 atm, respectively. Finally, a 10 ns MD simulation under the NVT ensemble was carried out with an integration time step of 1 fs, based on the periodic boundary condition. In the long time MD simulations, the SHAKE algorithm51 was applied to constrain all hydrogen-containing bonds with a tolerance of 10−5, and a 12 Å cutoff was set for both van der Waals and electrostatic interactions. The last 3 ns trajectories have been used for the root mean square fluctuation (RMSF) estimation and the binding free energy analysis. The final snapshot from the stable

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trajectory was used to prepare for subsequent QM/MM MD simulations. All classical MD simulations were accomplished by using the AMBER12 software.52 In order to figure out possible substrate delivery channels to the active site, the combined

random

acceleration

molecular

dynamics

and

MD

simulations

(RAMD-MD)53-55 have been carried out by using NAMD 2.9 software.50 Herein, the random accelerations of 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30 kcal Å−1 g−1 were added to the substrate and all these combinations above were repeated five times in RAMD simulations. Finally, 40 RAMD-MD trajectories for each model were identified. Based on the most probable channel determined from RAMD-MD simulations, the distance between the C1′ atom of ribose and the Ca2+ ion was chosen as the reaction coordinate (RC) for the substrate transportation, which varies from 18.0 Å to 4.1 Å for uridine and from 20.0 Å to 5.0 Å for inosine, respectively. Then the classical MD simulations combined with the umbrella sampling were carried out to get the free-energy profile for all windows separated by 0.4 Å. At least 8 ns MD simulations with the appropriate biasing harmonic potential (5~45 kcal/mol) along the path of the substrate delivery were carried out for each window. The last 5 ns reaction coordinate data for all windows were analyzed by the weighted histogram analysis method (WHAM) to generate the potential of mean force (PMF). QM/MM MD Simulations Each QM/MM model was prepared by deleting the solvent molecules beyond 30 Å from the Ca2+ ion, based on the equilibrated enzyme-substrate system from the classical MD simulations. The resulting QM/MM system consists of ∼16000 atoms. The QM subsystem consists of 51 atoms, including Asp11, Asp240, Ca2+, water molecules within 6 Å around Ca2+, and substrate in the active site of enzyme, which are directly involved in the breaking and formation of a C−N bond. The QM region is treated by using the B3LYP functional and the 6-31G* basis set. The rest of the protein and solvent is described by using the atomic force fields at the MM level, which is the same as that used in the above classical MD simulations. The QM/MM boundary was described by the improved pseudo-bond approach.56-58 The spherical boundary condition was applied, in which the atoms more than 30 Å away from the spherical center were fixed. The 18 and 12 Å cutoffs were employed for electrostatic and van der Waals interactions, respectively. There was no cutoff for electrostatic 5



interactions between QM and MM regions. In the QM/MM calculation, d1-r1-r2 was chosen as the reaction coordinate for all the models (refer to Figure 2), and the minimum energy path (MEP) for the catalytic step was mapped out by the reaction coordinate driving method,59 based on the QM/MM minimization calculation. Then, a 500ps MD simulation was utilized to equilibrate the MM part with the QM subsystem constrained. Finally, the ab initio QM/MM MD simulations combined with the umbrella sampling were carried out, and a constraint force constant of 10 ~ 40 kcal/mol/Å2 was imposed in MD simulations for all windows along the reaction coordinate. Each window was simulated for 20 ps with 1 fs time step at the QM(B3LYP/6-31G*)/MM level. The configurations from the last 5, 10, and 20 ps trajectories for each window were collected for data analysis. The probability distributions along the reaction coordinate were determined for each window and pieced together with the WHAM60-62 to calculate the potential of mean force (PMF). All our ab initio QM/MM calculations were performed with modified Q-Chem63 and Tinker64 programs.

Figure 2. The schematic diagram of the enzyme–substrate (uridine) complex. The black box refers to the active site of Yeik, and d1-r1-r2 was chosen as the reaction coordinate in the QM/MM calculation. 3. Results and discussion 3.1 The active-site structure and its binding interactions with the substrate The YeiK monomer comprises 11 α-helices, 10 β-strands, and a Ca2+ ion at the bottom of pocket.14 Owing to the missing of two flexible regions of the β3-α3 (loop1) and the C-terminal end of helix α9 (loop2) in the initial crystal structure of the 6


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enzyme-inhibitor complex (PDB ID: 3MKN), a repaired structure of the complex was constructed, and a 10 ns MD simulation was carried out to prepare an equilibrated configuration of the solvated enzyme-substrate complex. In Figure 3, the superimposition of the crystal and MD structures shows a high similarity between these two structures except two repaired flexible loops. The nucleophilic water molecule (w1) is 3.1 Å far from the C1’ atom of substrate, like all NH active sites, and the hydrogen bond interaction between the Ca-bound w1 and Asp11 was identified. Interestingly, three water molecules (w2, w3, and w4 in Figure 3) enter into the active domain after a 10 ns MD simulation, which may be involved in the leaving of groups.

Figure 3. Superimposition of the static crystal structure (PDB ID: 3MKN, green) of YeiK with an inhibitor (purple) and the MD-equilibrated configuration (gray) of YeiK with the substrate uridine (orange), where the Ca2+ ion is denoted as a cyan ball. Since both the anti- and syn-substrate conformations in Figure 4a are likely involved in the enzymatic catalysis, two corresponding models were prepared for analysis of the enzyme-substrate binding interactions. As shown in Figure 4 (b1)-(b2), the binding models after 10 ns MD simulations between the protein and anti- or syn-substrate are different from each other, in which the strong hydrogen-bond interactions between the base and Gln227 disappear in the syn-uridine system. Accordingly, there are relatively strong binding interactions between the anti-uridine substrate and the active-site residues, and the estimated binding free energies (∆G) of the uridine substrate are −45.7 and −29.3 kcal/mol for the anti- and syn-substrate conformations, respectively. Clearly, the conformation of the base moiety has remarkable effect on the substrate binding, while the binding pattern of the ribosyl group at the active site is basically conserved in the majority of NHs. The active site 7



of the Escherichia coli YeiK protein as a pyrimidine-specific NH prefers to accommodate the anti-uridine substrate in hydrolysis of the N-glycosidic bond, and thus the more stable anti-uridine conformation has been employed in the following investigation on the enzymatic hydrolysis of the N-glycosidic bond by CU-NH.

Figure 4. The chemical structure of syn- and anti-conformation (a). The structures of the active domain and its interactions with the uridine substrate in the syn-conformation (b1) and the anti-conformation (b2), where the red dash lines denote the hydrogen-bond interactions. 3.2 Catalytic mechanisms and effect of the substrate protonation In order to figure out the enzymatic reaction of CU-NH, possible hydrolysis mechanisms have been investigated by ab initio QM/MM MD simulations in combination with the umbrella sampling.32 The minimum energy pathways (MEPs) for the catalytic hydrolysis processes are predicted by QM/MM calculations, based on the reaction coordinate driving method.59 Herein, d1-r1-r2 (refer to Figure 2) is chosen as the reaction coordinate, which is similar to the choice in our previous study on the enzymatic hydrolysis by IAG-NH.35 8


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According to the determined MEPs, the potential of mean forces (PMFs) along the defined reaction coordinate have been calculated, and the relative free energy profiles and selected key structures during the hydrolysis are shown in Figure 5a. The PMFs from different periods of time are collected in Figure S2 in the Supporting Information, which suggest that the PMF profiles are well converged. The predicted free energy barrier for CU-NH is around 22.1 kcal/mol by QM(B3LYP/6-31G*)/MM MD simulations in combination with the umbrella sampling technique, which is reduced to about 20 kcal/mol after use of a relatively large 6-311+G** basis set for the QM region, based on the dual-level QM/MM approach

65-67

. The present results

show reasonable agreement with the approximatively estimated overall free energy barrier of 17.4 kcal/mol from the experimental kinetic data.11-12, 14

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Figure 5. The relative free energy profiles and the selected representative structures (a) along the hydrolysis reaction. Evolution of selected interatomic distances along the cleavage of N-glycosidic bond (b). As Figure 5 (a)-(b) shows, the enzymatic hydrolysis of uridine by CU-NH is a concerted and nonsynchronous process, accompanying with one proton transfer from the water molecule to the residue Asp11 during the cleavage of N-glycosidic bond. At the initial state, the oxygen atom of the reactant water molecule is 3.2 Å away from the C1′ atom of substrate, and its nucleophilic attack on the C1′ atom triggers the chemical step in hydrolysis of the N-glycosidic bond. As the water molecule approaches the C1′ site, the glycosidic bond length of N1−C1′ increases synchronously (see Figure 5b), and then the transition state is formed, along with conversion of the orbital hybridization from sp3 at the reactant state to sp2 for the C1′ atom of the substrate. As the reaction progress, the base leaves away from the ribose group further, and the oxygen of water attacks the C1′ atom by degrees. Finally, the hydroxyl group bonds to C1′, coupled with one proton transfer from the water molecule to Asp11. As shown in Figure 5b, the cleavage of N-glycosidic bond by CU-NH follows a highly dissociative and concerted mechanism, like the enzymatic hydrolysis by IAG-NH. Protonation of the nucleobase is believed to be important for the enzymatic hydrolysis by NHs. For the IU-NH from C. fasciculata, His241 was proposed as the proton donor to activate the hypoxanthine leaving group.68 In the kinetic isotope effect study on the uridine hydrolysis by the E. coli protein, it was assumed that the pyrimidine nucleobase might be activated by the hydrogen bonding interactions between the base and the enzyme or the substrate protonation.69 Our previous QM/MM MD simulations on the enzymatic hydrolysis of inosine by IAG-NH show that the substrate protonation at the N7 can reduce the free energy barrier remarkably and promote the cleavage of C1′-N bond.35 Presumably, the hydrogen bonding interactions between the enzyme and the substrate may play an important role in the substrate protonation. As shown in Figure 6, the active-site residues Gln227, Tyr231, and His239 in the initial configuration of the YeiK-uridine complex are probably to act as the proton donor for the substrate protonation. Based on the hydrogen bonding analyses of the 10


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last 5 ns MD simulation, Tyr231 always interacts with the O4 carbonyl oxygen of uracil ring via a strong hydrogen bond with the O•••O distance of 2.67±0.11 Å, and the O2 carbonyl oxygen of uracil ring also maintains a high probability of 89% to form the hydrogen bond with Gln227. However, it is basically impossible to form a stable hydrogen bond between the His239 and the N3 of uracil ring (2.66%). Owing to that the lone pair of N3 is involved in the ring conjugation interaction, the protonation at N3 requires more energy than those at O2 and O4 sites by 18.8 and 37.6 kcal/mol, respectively. Accordingly, the N3 protonation can be ruled out.

Figure 6. The probability of hydrogen bonds around the uridine substrate In order to evaluate the protonation effect on the N-glycosidic bond cleavage, the QM/MM energy scanning along the defined reaction coordinate for the possible protonated models have been performed and Figure 7 collects the relative energy profiles. The energy-scanning calculations indicate that the barriers are 26.0 and 22.0 kcal/mol for the N-glycosidic bond cleavage of the protonated substrate at O2 and O4, respectively. Obviously, for the uridine hydrolysis by CU-NH, the substrate protonation is unable to reduce the energy barrier for activation of the leaving group, quite differing from other NHs. Accordingly, the highly homologous CU-NH and IU-NH, belonging to Group I, have different pH dependences and working strategies in enzymatic hydrolysis of the N-glycosidic bond.

11



Figure 7. QM/MM-predicted relative energy profiles for the N-glycosidic bond cleavage in various protonated models of uridine. It was noted that the cleavage of C1′-N bond in the substrate of uridine initially yields a negatively-charged base group, and this intermediate configuration of product is about 17.0 kcal/mol higher in energy than the initial reactant state. The snapshots from our MD simulations reveal that there are side-chain residues, such as His82 and His239 in the release channel (refer to Figure 8), around the newly-generated negatively-charged base, and thus the leaving base group can easily accommodate one proton to form a more stable product of base. Our primary QM/MM calculations show that the proton transfer from the Asp11 to the negatively-charged base may release an energy of 5.5 kcal/mol. Furthermore, the strong solvation interactions between the base and the outer solvent water may stabilize the product of base and provide the driving force for the enzymatic hydrolysis.

Figure 8. A snapshot for the configuration during leaving of the base moiety. 3.3 Substrate Specificity It was well known that the IU-NH from C. fasciculata is a nonspecific hydrolase, suggesting that the common ribosyl moiety with three hydroxyl groups of substrate is 12


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crucial to the substrate binding and hydrolysis. However, the situation for the pyrimidine-specific CU-NH is more complicated, and selective binding interactions between the active-site residues and the base play a key role in manipulation of the substrate specificity.32, 70-72 The predicted binding free energy for inosine in the active site of the CU-NH Yeik enzyme is -28.3 kcal/mol, which is much smaller than that for the substrate uridine (-45.7 kcal/mol). The decrease of the binding free energy for inosine can be ascribed to the disappearance of the strong hydrogen-bond interactions between the protein environment and the base moiety as shown in Figures 9 and 4(b2), suggesting that the pyrimidine-specific CU-NH has a structure-selective active site toward the base moiety of substrate.

Figure 9. The hydrogen bond network around inosine in the active site of the CU-NH enzyme. The steady-state kinetic parameters for the pyrimidine-specific CU-NH YeiK indicate that its catalytic efficiency toward inosine, adenosine, or guanosine is at least 1000 times slower than that toward uridine.14, 73 Since such substrates have a common ribosyl moiety as shown in Figure 1, we may assume that the different base groups of substrates could be responsible for the substrate reactivity principally. The energy contribution of the main residues to the substrate-protein binding interactions is depicted in Figure 10a, which also suggests that both Gln227 and Asp15 are important for the substrate specificity of YeiK toward uridine. Computational analyses on the mutation of selected residues reveal that the Gln227/Ala mutation of the YeiK remarkably reduces the binding stability of uridine (The estimated binding free energy decreases from -45.7 to -35.3 kcal/mol) while slightly enhances the binding stability of inosine (The estimated binding free energies increase from -28.3 to -34.3 kcal/mol). 13



In addition, the Asp15/Ala mutant results in the binding free energies of -27.5 kcal/mol for uridine and -29.4 kcal/mol for inosine. Clearly, both residues Gln227 and Asp15 play a more notable role in the uridine binding, compared to the substrate inosine. As Figure 4(b2) shows, the residues Gln227 and Asp15 have direct hydrogen-bond interactions with the substrate uridine. On the contrary, the similar phenomenon was not found for the substrate inosine, and the binding interactions between inosine and the active-site residues of YeiK are less influenced by both Gln227/Ala and Asp15/Ala mutations. Presumably, these active-site residues of CU-NH YeiK may participate in encoding of the substrate specificity via specific enzyme-substrate interactions. Figure 10b collects the structural fluctuations of the CU-NH YeiK enzyme at the apo state and complex states with the substrates inosine and uridine, respectively. We note that the most remarkable decrease of RMSF appears as the uridine is bound to the active domain of the enzyme, while the RMSF values for the inosine binding are less changed or increased slightly in the partial region of protein relative to the apo state. These results also lend support to stronger interactions between uridine and the active-site residues of CU-NH YeiK, compared to inosine. We also estimated the volume74-75 of the active-site pocket of NHs from the last 5 ns MD simulation. After binding the substrate uridine, the active-site pocket of CU-NH is expanded slightly with the volume from 77.6 to 115.2±8.8 Å3. In comparison with CU-NH, the apo state of IU-NH from Crithidia has a larger binding pocket with the volume of 99.9 Å3. Furthermore, the binding of uridine to the IU-NH enzyme results in a remarkable expansion of its active-site pocket from 99.9 to 193.4±16.5 Å3, suggesting that IU-NH has a more flexible active-site pocket to accommodate a relatively big substrate, such as inosine. As observed in experiment,13 the IU-NH enzyme shows high activity toward the substrates both uridine and inosine. The predicted relative energy profiles for the enzymatic hydrolysis of N-glycosidic bond by QM/MM calculations are shown in Figure 11, and the estimated energy barriers are 22.1 and 30.3 kcal/mol for the substrates uridine and inosine, respectively, indicating that the enzymatic hydrolysis of inosine by CU-NH is much more difficult than that of uridine as observed experimentally.14, 22 Accordingly, the local active-site environment and its interactions with the substrate influence not only

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the substrate binding but also the chemical step involving the bond breaking and formation. Such nonchemical and chemical synergy will be responsible for the substrate specificity of CU-NH.

Figure 10. Comparison of the energy contribution from selected active-site residues of YeiK to the binding of substrates uridine (black) and inosine (red) (a). The RMSF of the protein backbone for the apo state (gray), the enzyme- inosine complex (red), and the enzyme-uridine complex (green), where the histograms refer to two flexible loop1 and loop2 (b).

15



Figure 11. QM/MM-predicted relative energy profiles for the N-glycosidic bond cleavage of substrates uridine and inosine. 3.4 Movement of the flexible loops As mentioned above, the flexible loop1 and loop2, located at the top of the active site, have not been resolved totally in the X-ray structure, and they may remarkably influence the structure of the active site. As Figure 12a shows, there are two relatively large RMSF peaks highlighted by the blue histograms, which correspond to loop1 and loop2, respectively. These two loops are more flexible than the rest of protein as expected, and they undergo a large change in the apo state of CU-NH. Once the uridine substrate bound to the active site, the enzyme-substrate complex, including the loop1 and loop2, becomes more ordered, especially for loop2. Actually, two distinct conformations, namely the open and closed forms, have been characterized in previous X-ray diffraction and the free-energy profiles of the transition between two endpoints.23 Clearly, such conformational change can be ascribed to movement of the flexible loop1 and loop2. As Figures 12 and 2 show, the loop 1 (amino acids 72–97), acting as a “lid”, is located on the top of the active site, and there are relatively weak van der Waals interactions between the pyrimidine ring and the side-chain residues Ala78, Ile81, and His82 of loop1 (see Figure S3). Besides, the unresolved C-terminal residues of helix 9 (amino acids 216–236 of loop2) in the crystal structure become much less flexible in the presence of substrate and fold to α-helix (refer to Figure S3). This conformational change basically arises from the strong hydrogen bond interactions between the active-site residues Gln227 and Tyr231 in the loop2 and the

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uridine substrate.

Figure 12. The root mean square fluctuation (RMSF) of the protein backbone for the apo state (black) and the enzyme-uridine complex (red), in which the histograms correspond to two flexible loop1 and loop2 (a). Energy contribution of the main residues to the substrate-protein binding interaction (b).

17



Figure 13. The possible channels for uridine and inosine delivery to the active site predicted by RAMD-MD simulations. 3.5 Substrate Delivery to the Active Site For the substrate binding, two possible channels, defined as a1 and b1, were detected, which are displayed in Figure 13. The path a1, located between the flexible loop 1 and loop 2, has a dominant share of trajectories (~72.5% for uridine and ~92.5% for inosine). Only 7 and 3 trajectories arise from the path b1 for uridine and inosine, respectively, among 40 trajectories. Path b1 is located between loop 2 and helix 8. As Figure 14 shows, the uridine binding is a remarkable exothermic (~23 kcal/mol) and almost barrier-free (~1 kcal/mol) process, which is much more favorable thermodynamically than the inosone binding with an energy release of ~12 kcal/mol and a barrier of ~3 kcal/mol. Such differences in the transport behavior of substrate are also responsible for the substrate specificity.

Figure 14. The predicted free energy profiles for the access of uridine and inosine to the active site. 18


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Based on the conformational changes, the uridine transport to the active site can be approximately divided into three stages (see Figure S4 in the Supporting Information). In the first stage (RC > 11.5 Å), the residue Tyr231, as the leader of loop 2, maintains an open state to let the uridine substrate enter into the active site. We note that the pocket is water-filled and the loop 2 is quite flexible. In the second stage (7 < RC < 11.5 Å), the substrate will pass through the residue Tyr231. With the substrate entrance, the hydrogen bond between the residue Tyr231 and uridine is formed, which makes loop 2 become more stable α helix. Similarly, the hydrogen-bond interactions between the substrate and loop 1 appear gradually, which may facilitate the substrate binding. In the third stage (RC < 7 Å), many water molecules are extruded from the pocket (see Figure S5 in the Supporting Information), and strong hydrogen-bond interactions of uridine with the residues Tyr231 and Gln227 from loop 2 are formed. Only one water molecule is involved in the interactions of the octacoordinated calcium ion with protein, which will be responsible for the nucleophilic attacking of hydrolysis. 4. Conclusions Extensive MM MD and QM/MM MD simulations have been used to explore the enzymatic hydrolysis of uridine by CU-NH, and the structural features of active domain, loop motion, substrate specificity, substrate delivery, and plausible catalytic mechanisms have been discussed. Current calculations and simulations indicate that the anti-uridine substrate has stronger binding interactions with the active-site residues, compared to the syn-uridine, and it as a favorable configuration is involved in the enzymatic hydrolysis. Once the substrate bound to the active site, the flexible loop 1 and loop 2, as well as the protein backbone become more ordered, and the hydrogen-bond interactions between the substrate and the side-chain residues of loops may contribute the substrate specificity to some extent, although it is basically dominated by the kinetic barriers. Plausible transportation pathways of the substrate to the active site have been identified by using RAMD-MD simulations, and the conformational change of the residue Tyr231 of loop 2 as well as noncovalent interactions between the substrate and the side-chain residues play an important role for the substrate binding. The access of uridine to the active site is predicted to be much more favorable thermodynamically, compared to the inosine substrate. Here the enzymatic cleavage of the N-glycosidic bond by CU-NH follows a highly dissociative 19



concerted and nonsynchronous mechanism, and no activity enhancement from the substrate protonation has been found. The present results provide new insights into the enzymatic hydrolysis by pyrimidine nucleoside hydrolases.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: The three-dimensional structure of YeiK monomer, the tetrameric structure of YeiK, the predicted relative free energy profiles for the cleavage of N-glycosidic bond of uridine by QM/MM MD simulations from different time scales, the conformation of key residues from loop 1 in the apo state and the enzyme-substrate complex, the hydrogen-bond interactions between the substrate and key residues from loop 2, the key residues in the active site and their conformational changes during the uridine movement along the reaction coordinate, the evolution of number of water molecules in the active domain as the uridine approaches the active site.

Author Information Corresponding author *E-mail: [email protected]. Phone: +86-592-2186081. Notes The authors declare no competing financial interest. Acknowledgements This work was supported by the National Science Foundation of China (21373164 and 21673185).

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MM and MM MD Simulations on the Pyrimidine-Specific

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