New Delhi Metallo-β-Lactamase I: Substrate Binding and Catalytic

Aug 27, 2013 - José Rogério A. Silva , Thavendran Govender , Glenn E. M. Maguire , Hendrik G. Kruger , Jerônimo Lameira , Adrian E. Roitberg , Clá...
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New Delhi Metallo-β-Lactamase I: Substrate Binding and Catalytic Mechanism Min Zheng and Dingguo Xu* College of Chemistry, Sichuan University, Chengdu, Sichuan 610064, P. R. China S Supporting Information *

ABSTRACT: Metallo-β-lactamases can hydrolyze and deactivate lactamcontaining antibiotics, which is the major mechanism for causing drug resistance in the treatment of bacterial infections. This has become a global concern because of the lack of clinically approved inhibitors so far. The emergence of New Delhi metallo-β-lactamase I (NDM-1) makes the situation even more serious. In this work, first, the structure of NDM-1 in complex with the inhibitor molecule L-captopril is investigated by both density functional theory (DFT) and hybrid quantum mechanical/molecular mechanical (QM/ MM) methods, and the theoretical results are in good agreement with the Xray structure. The Michaelis structure with an antibiotic compound (ampicillin) bound in the active site is constructed from a recent X-ray structure of the NDM-1 enzyme with hydrolyzed ampicillin. It is further simulated using a QM/MM molecular dynamics method. One of the interesting binding features of ampicillin in the NDM-1 active site is that the conserved C3 carboxylate group is not ligated with Zn2 but rather is only hydrogen-bonded with N220 and K211. A bridging hydroxide ion is suggested to connect two zinc cofactors. This hydroxide ion is also hydrogen-bonded with D124. Subsequent reaction path calculations indicate that the initial step of lactam ring-opening occurs through a concerted step in which the cleavage of the C−N bond and the transfer of the hydrogen bond to D124 are nearly concerted. The ligand bond between Zn2 and the C3 carboxylate group forms after the first step of nucleophilic addition. The calculated activation energy barrier height is about 19.4 kcal/mol for the hydrolysis of ampicillin, which can be compared with the experimental value of 15.8 kcal/mol derived from kcat = 15 s−1. The overall mechanism is finally confirmed by a subsequent DFT study of a truncated active-site model. application to nearly all β-lactam antibiotics, especially the occurrence of carbapenemases,6 makes them an increasing challenge to existing clinical therapy. The complete absence of clinically useful inhibitors further aggravates the current worry. This problem can be largely attributed to the lack of information about the structure of the Michaelis complex and corresponding catalytic mechanism. Very recently, a new member of the B1 class of metallo-βlactamases was found in a patient who traveled from India to Switzerland in 2008,7 which was named New Delhi metallo-βlactamase I (NDM-1). Clinically, NDM-1-bearing Klebsiella pneumonia and Escherichia coli were shown to be resistant to nearly all β-lactam antibiotics.7 These so-called “superbugs” represent a new round of health threats in treating bacterial infections. Considerable efforts have been devoted to understanding the three-dimensional structure8−13 and catalytic mechanism7,14,15 of NDM-1. However, the number of zinc ions binding in the active site is still controversial. A recent Xray structure of NDM-1 in complex with a hydrolyzed ampicillin molecule reported by Zhang and Hao9 clearly

1. INTRODUCTION It is very hard to imagine a world without effective antibiotics because of their essential roles in the treatment of various bacterial infections. Since the introduction of penicillin by Sir Alexander Fleming in 1928, bacteria have evolved to develop powerful weapons against those antibiotics, such as βlactamases. This has become a major cause of worldwide health concern for bacterial resistance.1,2 β-Lactamases can hydrolyze β-lactam-containing antibiotics, causing the loss of their activity in clinical therapy. On the basis of the amino sequence and catalytic mechanism, β-lactamases can be classified into four classes. Among them, classes A, C, and D are serine-based β-lactamases, which catalyze the hydrolysis of β-lactam antibiotics through a covalent mechanism.3 The members of class B, on the other hand, require one or two zinc ions in the active site to maintain their enzyme activity and, thus, are called metallo-β-lactamases (MβLs). The B class can be further divided into three subclasses, B1−B3.4 Generally, B1 and B3 β-lactamases can accommodate two zinc cofactors in the active site, whereas B2 is a monozinc enzyme. Because of the diversity of structures, about 20% similarity among MβLs, different substrate binding patterns, and catalytic mechanisms can be observed.5 Although discovered not long ago in quite a few populations around the world, their broad spectrum © XXXX American Chemical Society

Received: July 3, 2013 Revised: August 20, 2013

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Scheme 1. Atom Definitions and Corresponding Interactions between L-Captopril/Ampicilin and the Active-Site Residues of NDM-1

polar residues near Zn2 such as either lysine or serine have been suggested to be important for the recognition of lactam substrates. In addition, based their new reported structure, Kim et al. further proposed a new mechanism in which the bridged hydroxide plays the role of general base to activate a putative active-site water molecule. A key point is that no anionic nitrogen intermediate occurs in their reaction process. These conclusions are clearly very different from those of previous theoretical and experimental studies, and thus, an alternative mechanism and substrate binding mode might be expected. On the other hand, Kim et al.13 also admitted that the fourmembered ring of unhydrolyzed ampicillin substrate of their reported structure is disordered because the electron density for the substrate molecule seems to be different from those of normal hydrolyzed ampicillin and unhydrolyzed ampicillin. Regarding this point, some more explanations for the electron density are thus highly required. After all, incorrect interpretations of the X-ray data might cause misleading conclusions about both the structure and mechanism.20,21 According to the above discussion, the Kim et al.13 model for the initial Michaelis complex should be questioned, and some higher-resolution X-ray structural analysis or more theoretical simulations should be performed. Given the controversial understanding of the substrate binding mode and catalytic mechanism for NDM-1, extensive investigations at the molecular level are highly required. In this work, we address the binding of unhydrolyzed ampicillin to the NDM-1 active site and the corresponding cleavage of the amide bond of the β-lactam ring using both the QM/MM method and density functional theory.

indicates the presence of two zinc ions in the active site. It is now clear that the two zinc ions have different binding environments. For the first metal binding position (Zn1), the zinc ion is surrounded by three histidine residues, H120, H122, and H189. For the second metal binding position (Zn2), the zinc ion is ligated by D124, C208, and H250. When the substrate is bound in the active site, the fourth ligand to zinc ions is believed to be a hydroxide ion, which stays almost in the middle of the two zinc ions. A later extensive analysis also suggested that the binding affinities of the two zinc cofactors are different, with the binding affinity of Zn1 being stronger than that of Zn2.14 However, it should be noted that the inclusion of Zn2 could significantly increase the catalytic activity, which highlights the importance of Zn2 in the hydrolytic reaction. In a recent hybrid quantum mechanical and molecular mechanical (QM/MM)16 molecular dynamics (MD) simulation of apo NDM-1,14 the structural similarity with another B1 β-lactamase, VIM-4, was established. The recent mechanistic and spectroscopic studies of NDM-1 suggested that NDM-1 utilizes a kinetic mechanism similar to that used by other MβLs such as CcrA found in Bacteroides f ragilis.15 Based on the X-ray structure, Zhang and Hao9 proposed a general mechanism for the hydrolysis of ampicillin. Later, Crowder and co-workers15 suggested that the formation of an anionic nitrogen intermediate and protonation of nitrogen anion should be the major steps for NDM-1 catalysis. Such an intermediate is a common feature for the β-lactam antibiotics hydrolysis catalyzed by MβLs. Indeed, a similar anionic nitrogen intermediate was identified in previous theoretical simulations of other MβLs.17,18 In one recent work involving combined X-ray structure and QM/MM investigations of the substrate recognition and catalytic mechanism for NDM-1, Kim et al.13 proposed that the broad spectrum against all lactam antibiotics of NDM-1 might come from a special binding pattern, in which the only interaction between the substrate and the protein is that of the C3 carboxylate group with Zn2. Another important binding feature is that there is absolutely no connection between unhydrolyzed ampicillin and protein residues. This observation might be questioned, because the recent site-directed mutagenesis study of the nearby lysine residue of K211 might completely deactivate the NDM-1.19 This could partially indicate that K211 plays some key roles in the catalysis and substrate binding. Meanwhile, as shown in the X-ray structure reported by Zhang and Hao9 and as observed for other MβLs,

2. COMPUTATIONAL DETAILS 2.1. QM/MM Model. As reviewed in many previous articles,22−24 the QM/MM method is widely considered as a powerful tool for understanding complicated processes occurring in extended systems such as enzymes. The basic idea is to divide the overall system into two parts. The smaller region includes all of the atoms involved in the reaction and is called as QM region. The remainder (MM) includes the protein environment and solvent molecules, which is then described using a force-field method. It has long been recognized that the correct selection of a high-level quantum mechanical method in QM/MM simulations is needed for an accurate understanding of the final results. Ideally, it would be better to utilize either ab initio methods or density functional theory in calculations of the B

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2.2. DFT Model. To independently examine the enzymatic reactive mechanism obtained by the combined SCC-DFTB/ MM approach, we carried out a noncatalyzed hydrolysis reaction simulation on a truncated active-site model using the density functional theory (DFT) method. The truncated activesite model for the reactant complex (RC) was adopted using the optimized QM region from the overall ES complex system, in which the histidine and aspartic acid residues were simulated using methyl imidazole and acetate molecules, respectively. Methylamine was used to approximate the K211 residue, whereas the 3-(mercapto)propionaldehyde analogue was taken to replace the C208 residue. In addition, the DFT model also includes the full ampicillin, two zinc ions, and the bridging hydroxide ion. The Becke three-parameter Lee−Yang−Parr (B3LYP) exchange-correlation functional40,41 with a standard basis set [6-31G(d)] was employed in our computations. Full geometry optimizations without any constraints were carried out for all stationary states, which were further confirmed by harmonic vibrational frequency calculations, with only one imaginary frequency for the transition state (TS) and all positive frequencies for the minima [RC and intermediate complex (INT)]. Intrinsic reactant coordinate (IRC) calculations were finally applied to connect all of the stationary structures.42 All DFT calculations were performed using the Gaussian 09 suite of programs.43

electronic structure of the QM region. However, the application of such approaches is still restricted by their high computational costs. In this work, the self-consistent charge-density functional tight binding (SCC-DFTB)25,26 method combined with the CHARMM2227 all-atom force field was used in all of the QM/ MM simulations. The SCC-DFTB method was incorporated into the CHARMM software package in 2001.28 In particular, to correctly simulate the physiological features of biological zinc cofactors, the parameters for the SCC-DFTB method were also reported.29 Since then, this method has been successfully applied to investigate several important zinc-containing enzymes, including carbonic anhydrases,30,31 amino peptidases,18,32,33 and metallo-β- lactamases.18,34,35 The initial structure was extracted from the X-ray structure of chain B of NDM-1 in complex with a hydrolyzed ampicillin molecule [Protein Data Bank (PDB) code 3Q6X].9 The native ampicillin molecule was then recovered by manually replacing the C7 carboxylate group with a carbonyl group and reconnecting the amide C−N bond to form the lactam ring. In the X-ray structure, a bridging oxygen atom exists between the two zinc ions. It is simulated as a hydroxide ion considering the effects from the zinc ions, as has been proposed for other dizinc β-lactamases.14,36 This hydroxide ion acted as the putative nucleophile in the investigations of substrate binding pattern and catalytic mechanism. Hydrogen atoms were carefully added to the heavy atoms according to their environment using HBUILD module. Possible interactions around the active site and atom definitions are given in Scheme 1. One of major differences between the X-ray structure and our putative substrate binding pattern is the ligation status of the C3 carboxylate group. Unlike in the X-ray structure and other dizinc β-lactamases, it is not the fourth ligand of Zn2 but, rather, hydrogen-bonded with K211. We will discuss this binding feature in more detail in the following sections. To correctly include all of the reactive information, the QM region thus consists of the ampicillin molecule; bridging hydroxide ion; side-chain group K211; two zinc ions; and sidechain groups of their ligands of H120, H122, H189, D124, C208, and H250. The total number of atoms in the QM region is thus 121, and the total charge of the QM system is +2. After the enzyme−substrate (ES) complex was constructed, it was solvated by a pre-equilibrated TIP3P37 water sphere of 25-Å radius centered at the carbonyl carbon (C7) of the lactam ring. All water molecules within a 2.8-Å radius of a heavy atom were deleted. This process was repeated several times with rotated water spheres to ensure uniform solvation. The solvent molecules were further relaxed with a 30-ps molecular dynamics (MD) simulation in which all protein atoms, the substrate molecule, and the zinc ions fixed at their original positions. Stochastic boundary conditions38 were applied to reduce the computational cost. Atoms 25 Å from the origin were removed. The overall system contained a total of 7112 atoms, including 366 water molecules. The constructed system was first minimized using the steepest-descent (SD) method followed by the adopted basis Newton−Raphson (ABNR) method. The optimized complex system was subjected to a 1.5-ns MD simulation. The system was first slowly heated to 300 K within 30 ps, and the subsequent 470 ps was used for further equilibration. The rest of the 1-ns MD trajectory was saved for product data analysis. The integration time step was 1.0 fs, and the SHAKE algorithm39 was applied to maintain the covalent bonds involving hydrogen atoms.

3. RESULTS AND DISCUSSION 3.1. NDM-1/L-Captopril Complex. Before investigating the native substrate binding pattern of NDM-1, it is necessary to validate the combined SCC-DFTB/MM approach in the study of the current enzyme system. We have applied SCCDFTB/CHARMM in the simulation of apo MβLs and substrate binding patterns for CphA from Aeromonas hydrophila44 and L1 from Stenotrophomonas maltophilia.34 For the performance with a small molecule in the NDM-1 active site, we selected its specific inhibitor, L-captopril, for which the crystallographic structure of NDM-1 in complex with the inhibitor was recently reported.12 This can provide a very good starting point for subsequent MD simulations. L-Captopril, combined with D-captopril, displays potent and broad-spectrum MβL inhibitory activity.45 The setup protocol was essentially the same as described above for the ampicillin binding simulations. The initial structure was taken from the X-ray structure of chain A of NDM-1 in complex with the inhibitor, L-captopril (PDB code 4EXS).12 As shown in Scheme 1, the sulfur atom of L-captopril stays between the two zinc ions to be the fourth ligand of the two zinc ions. A 1.29-ns MD simulation was applied to investigate the performance of captopril in the active site. The first 290 ps of the simulation was used for slow heating to room temperature (300 K) and system equilibration. The rest of the 1-ns MD trajectory was then saved for further data analysis. The overall structure was found to be quite stable, as evidenced by the root-mean-square deviation (RMSD) of 1.18 ± 0.09 Å for the backbone atoms (see Figure 1). An overlap representation between the X-ray structure and a snapshot from the trajectory is given in Figure 2A, and selected key geometric parameters are listed in Table 1. Throughout the simulation, the ligation status between the sulfur atom of Lcaptopril and the two zinc ions was well-maintained, as judged by the distances of 2.37 ± 0.07 Å for S−Zn1 and 2.41 ± 0.08 Å for S−Zn2. Meanwhile, the zinc ion at the Zn1 binding position was coordinated with H120, H122, and H189. The C

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Table 1. Key Distances (Å) of NDM-1 in Complex with LCaptopril Obtained Using SCC-DFTB/CHARMM and DFT Truncated Model Calculations, along with the X-ray Structure Parameters for Comparison distance (Å) Zn1···S1 Zn1···Zn2 Zn2··· S1 Zn1···Nε2(His189) Zn1···Nδ1(His122) Zn1···Nε2(His120) Zn2···Nε2(His250) Zn2···Oδ2(Asp124) Zn2···S(Cys208) S1···Oδ1(Asp124)

Figure 1. RMSDs of both enzyme−inhibitor complex (blue line) and enzyme−substrate complex (red line) as a function of time.

corresponding differences in the ligand distances were typically less than 0.05 Å compared with the experimental distances. Similar results were observed for the ligation status of Zn2. Because of the lack of a C3 carboxylate group in the inhibitor molecule, K211 could not form a hydrogen bond with the inhibitor molecule, but instead stabilized one of the zinc ligands, C208. Because the side-chain amino group of K211 rotated during the simulation, we cannot provide a statistically average value for the distance between a specific hydrogen atom of the amino group and the backbone oxygen atom of C208. The Nζ(K211)···O(C208) distance was calculated to be 2.81 ± 0.14 Å, which agrees well with the X-ray value of 2.92 Å. The conserved asparagine residue, N220, has been suggested to be important in the binding of various substrates on the basis of either experimental observations or theoretical simulations.46,47 For the recognition of L-captopril, it has been suggested that its carboxylate group is anchored by the backbone amide group of N220, with distances as long as 3.3 Å between the O2 atom of L-captopril and the amide group and N220. However, such long distances cannot ensure a strong possible hydrogen bond. Indeed, in our simulations, this interaction was quickly lost, and the terminal carboxylate group of L-captopril was completely exposed to the solvent environment. Interestingly, for the binding of biapenem to the CphA active site,44,46 the side-chain amino group of N233 forms a hydrogen bond with the lactamring carbonyl oxygen atom, which can provide additional stabilization to the binding of the substrate. For the binding of L-captopril, there is no such interaction because of the lack of the carbonyl group. Without this hydrogen bond, the proline group of L-captopril seems to be quite flexible in the active site, as shown by the overlap representation in Figure 2A. With this exception, our MD simulations can largely reproduce the X-ray structure, as shown in Table 1. To independently validate the SCC-DFTB/MM approach in the simulation of NDM-1, we performed a high-level DFT optimization of a truncated active-site model at the B3LYP/631G(d) level of theory. To reduce the computational cost, only

SCC-DFTB/MM MD

DFT

X-ray12

± ± ± ± ± ± ± ± ± ±

2.35 4.00 2.43 2.06 2.04 2.02 2.08 1.97 2.30 3.65

2.28 3.59 2.32 2.03 2.08 2.08 2.18 1.92 2.13 3.80

2.37 3.70 2.41 2.01 2.00 2.01 2.04 2.06 2.34 3.80

0.07 0.16 0.08 0.05 0.06 0.06 0.06 0.06 0.07 0.22

the QM region was used in the simulation, with the terminal carboxylate group of L-captopril molecule simply replaced by a hydrogen atom. In addition, the histidine residue was simulated using methyl imidazole, acetate was used to approximate the aspartic acid residue, and 3-(mercapto)propionaldehyde was used to mimic C208. Because K211 does not provide additional stabilization for the binding of L-captopril, we did not include it in the DFT calculations. Some selected geometric parameters are also included in Table 1 for comparison, and the corresponding DFT-optimized structure is displayed in Figure 2B. In summary, the optimized truncated model obtained at a high level of theory was found to agree well with the geometries from the SCC-DFTB/MM MD simulations, although some deviations were observed because of the lack of solvent molecules and the protein environment. For example, the distances between the sulfur atom and the two zinc cofactors were calculated to be 2.35 Å for Zn1···S1 and 2.43 Å for Zn2··· S1, which are very close to the values from the QM/MM MD simulation of 2.37 ± 0.07 and 2.41 ± 0.08 Å, respectively. Such results provide reassurance for the application of the SCCDFTB/MM method to further simulations of the substrate binding pattern and possible enzymatic reaction mechanism. 3.2. Enzyme−Substrate Complex. As a member of a successful penicillin family of β-lactam antibiotics, ampicillin has been extensively used in the treatment of bacterial infections since 1961. Very recently, the crystallographic structure of NDM-1 in complex with hydrolyzed ampicillin at 1.3-Å resolution was reported by Zhang and Hao.9 Some important geometric information is revealed by the X-ray structure, such as the zinc binding motif and residues helping substrate binding. On the basis of this structure, Zhang and

Figure 2. (A) Overlap representation of NDM-1 in complex with L-captopril extracted from the MD trajectory (carbon atoms in green, zinc cofactors in purple) and the X-ray structure (carbon atoms in yellow, zinc ions in gray). (B) DFT-optimized active site of NDM-1 with an L-captopril molecule. D

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Hao proposed a model for substrate binding and a possible hydrolysis mechanism. In this work, we further carried out extensive QM/MM MD simulations of NDM-1 in complex with an unhydrolyzed ampicillin molecule. Indeed, information on the structure of the ES complex is more important for structure-based inhibitor design and understanding of the hydrolysis mechanism As mentioned above, for lactam-ring-containing antibiotics, there is a conserved C3/C4 carboxylate group. Therefore, the binding characteristics around this group are a key issue. As suggested by Zhang and Hao,9 the C3 carboxylate group of hydrolyzed ampicillin is one of the ligands of the Zn2 cofactor. This proposal was also widely accepted in previous theoretical simulations of other dizinc metallo-β-lactamases.5,34 In this work, we first built a model with the C3 carboxylate group ligated with Zn2. Surprisingly, the MD simulations quickly broke this ligation to convert Zn2 to a tetracoordination status. The model system used in the subsequent MD simulations was thus that shown in Scheme 1 with tetracoordination for Zn2. However, because the formation of a ligand bond between the C3 carboxylate group and Zn2 was observed for NDM-1 in complex with the hydrolyzed substrate, the question of how and when this ligand bond forms deserves special investigation. First, detailed analysis of ampicillin binding characteristics are presented. Throughout the simulations, ampicillin remains quite tightly in the active site. The overall structure is also quite stable, as evidenced by the RMSD of 0.74 ± 0.06 Å for backbone atoms compared with the X-ray structure, as shown in Figure 1. Some selected time-averaged geometric parameters are listed in Table 2, and a snapshot extracted from the MD trajectory is displayed in Figure 3. As in L-captopril binding, the two zinc ions are again tetracoordinated. Zn1 is surrounded by the three histidine residues H120, H122, and H189. Zn2 is ligated with D124, C208, and H250. The two zinc ions are separated by a distance

Figure 3. Snapshot of NDM-1 in complex with ampicillin. Black lines represent hydrogen bonds, and purple lines represent metal−ligand bonds.

of about 3.58 ± 0.11 Å, which seems to be a typical distance for dizinc MβLs.34,48,49 The bridged hydroxide ion is the fourth ligand of the two zinc ions and stays nearly in the middle of the two zinc ions, with distances of 2.03 ± 0.06 Å for Ow−Zn1 and 2.03 ± 0.06 Å for Ow−Zn2. Meanwhile, this hydroxide ion is further positioned by a hydrogen bond provided by the conserved aspartic acid residue of D124. The corresponding hydrogen-bond distance was calculated to be 1.92 ± 0.15 Å. The importance of this conserved aspartic acid residue in metalβ-lactamases has long been recognized in both simulations 14,34,48,50,51 and side-directed mutagenesis experiments.52,53 This residue has been suggested to function as the general base to activate the active-site water nucleophile35 or just the hydrogen acceptor17 to facilitate the further hydrolysis reaction in MβLs. This ligation status and corresponding hydrogen bond network of the bridged hydroxide ion are also a common feature among those dizinc MβLs.36 Additionally, the distance between the putative nucleophile (Ow atom) and carbonyl carbon atom C7 was averaged to be 2.96 ± 0.34 Å; C7 stays right below the lactam ring, as shown in Figure 3, and is ready for the subsequent nucleophilic addition reaction. Moreover, the binding of ampicillin stays in the core position for further discussion of reaction mechanism. It can also provide more detailed insight into effective inhibitor design. The binding feature of the conserved C3/C4 carboxylate group has been widely discussed, and many suggestions have been made. The generally accepted opinion is that Zn2 can directly interact with the carboxylate group to enhance the substrate binding in the active site.5 However, it should be pointed out that current experimental evidence for β-lactamases can support the conclusion only that the hydrolyzed intermediate or product has such a binding feature for the C3/C4 carboxylate group ligated with Zn2. No direct structural evidence has been revealed to support this interaction in the ES structure because of the highly efficient catalytic rate. On the other hand, parameters for biological zinc ions developed for SCC-DFTB can support pentacoordination, because we observed the pentacoordination of Zn2 in our previous simulations of the binding of moxalactam to B3 L1.34 Interestingly, unlike for moxalactam in the L1 active site49 and biapenem in CphA,46 there was no direct contact between the substrate and the zinc ion at the second binding position (Zn2) for NDM-1 in the current simulations. The C3 carboxylate group of ampicillin stays quite far from Zn2, with a value of 4.99 ± 0.37 Å for

Table 2. Key Distances (Å) of NDM-1 in Complex with Ampicillin Obtained Using the SCC-DFTB/MM and DFT Methods, along with the X-ray Structure Parameters of NDM-1 in Complex with Hydrolzed Ampicilin for Comparison distance

SCC-DFTB/MM MD

DFT

X-ray9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.91 3.58 3.98 1.98 2.03 2.04 2.01 2.07 1.98 2.30 5.14 5.32 1.75 1.60 2.52 1.82 -

2.02 4.59 2.95 2.99 2.04 1.99 2.11 2.02 2.15 2.35 2.18 2.18 2.48a 2.75b 3.17b 2.80b 3.07b 3.07b

Zn1···Ow Zn1···Zn2 Ow···C7 Zn2··· Ow Zn1···Nε2(His189) Zn1···Nδ1(His122) Zn1···Nε2(His120) Zn2···Nε2(His250) Zn2···Oδ2(Asp124) Zn2···S(Cys208) Zn2···N4 Zn2···O9 Hw···Oδ1(Asp124) Hζ1(Lys211)···O10 Hζ1(Lys211)···O9 Hζ2(Lys211)···O(Cys208) Hδ22(Asn220)···O8 HN(Asn220)···O10 a

2.03 3.58 2.96 2.03 2.01 2.01 2.04 2.03 2.12 2.32 3.94 4.99 1.92 2.52 1.39 2.15 2.13 1.90

0.06 0.11 0.34 0.06 0.05 0.05 0.06 0.06 0.07 0.06 0.32 0.37 0.15 0.16 0.07 0.54 0.35 0.24

O−O distance. bN−O distance. E

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Figure 4. (A) QM/MM minimum-energy surface and (B) corresponding minimum-energy curve along the reaction coordinate of d1 for the hydrolysis of ampicillin catalyzed by NDM-1. The B3LYP/MM//SCC-DFTB/MM single-point energy curve (blue line in panel B) along the SCCDFTB/MM calculated minimum-energy curve is also included for comparison.

hand, because we observed the formation of a ligand bond between the C3 carboxylate group and Zn2 in the X-ray structure of NDM-1 in complex with hydrolyzed ampicillin, we have to answer the question of when or how this ligand bond is formed. In the X-ray structure of NDM-1 in complex with hydrolyzed ampicillin, the amide nitrogen atom becomes one of the ligands of Zn2 with a distance of 2.18 Å because the nitrogen atom (N4) is negatively charged after the first step of the ringopening reaction. In contrast, there is no negative charge on the N4 atom for nonhydrolyzed ampicillin, so the N4 atom stays relatively far from Zn2, with a distance of 3.94 ± 0.32 Å. 3.3. Reaction Path Calculations. 3.3.1. Searching for the Reaction Coordinates. As mentioned above, the binding characteristics of the C3 carboxylate group for NDM-1 are different from those observed in our previous simulations of CphA and L1. To correctly understand this difference, extensive reaction simulations should be carried out to validate this proposal. In this work, we further present our calculations of the first step of lactam ring opening using the combined QM/MM approach. First, a correct definition of the reaction coordinates is a prerequisite to understanding the overall reaction process. Initially, we followed the reaction coordinate definition that we used for the first step of the hydrolysis of moxalactam catalyzed by L1,17 which includes nucleophilic addition by the hydroxide ion and cleavage of the C−N bond. To examine whether there is also a metastable intermediate related to the proton transfer from the hydroxide ion to D124, we then included the proton transfer in the reaction coordinates to calculate a twodimensional potential energy surface (PES) using the so-called adiabatic mapping approach (sometimes referred to as the reaction coordinate driving method). With the approach of Ow to C7, the distance between the C3 carboxylate group (O9) and Zn2 is decreased because of the attraction between negative and positive charges. As shown in Figure S1 (Supporting Information), the formation of the O9−Zn2 ligand bond could lead to a very sharp change in the potential energy surface (PES), so that the PES is not continuous and smooth. In addition, the direction of proton transfer is barrierless. These results obviously indicate that the formation of the O9−Zn2 ligand bond should be one of the important factors in the first step of the hydrolysis of ampicillin catalyzed by NDM-1.

dZn2−O9. Instead, two hydrogen bonds are provided from the enzyme environment to help stabilize the C3 carboxylate group. The major contribution to the stabilization of the C3 carboxylate group is provided by K211 through a salt bridge with a short distance of 1.39 ± 0.07 Å for Hζ1(K211)···O9. The hydrogen bond formed between the substrate carboxylate group and a polar residue seems to be a general substrate recognition characteristic for nearly all β-lactam antibiotics, for example, K211 for NDM-1, K167 for CcrA,54 K224 for CphA,46 R119 for VIM-4,55 and two serine residues (S221 and S223) for B3 L1.49 Moreover, this lysine residue is also connected to the backbone amide oxygen atom of C208, one of the zinc ion ligands, with a hydrogen-bond length of 2.15 ± 0.54 Å. The kinetic importance of K211 is further highlighted by two mutants of K211A and K211E, in which the enzyme activity is completely disrupted when the meropenem molecule is used as the substrate.19 Similarly to our previous studies of biapenem binding to B2 CphA,47 we also found that N220 plays a donor function to anchor the substrate molecule. Two hydrogen bonds formed between the substrate and N220 can be observed. The hydrogen bond [dHN(N220)···O10 = 1.90 ± 0.24 Å] between the C3 carboxylate group and the N220 backbone amide nitrogen atom can facilitate the stabilization of the ampicillin, whereas the hydrogen bond [dHδ22(N220)···O8 = 2.13 ± 0.35 Å] between N220 and the lactam-ring carbonyl oxygen atom could indicate that N220 has the function of oxyanion hole for the cleavage of the amide C−N bond through polarization. In a recent hybrid PM3/MM and DFT simulation of B1 CcrA in complex with nitrocefin molecule,48 a similar ligation system for two zinc ions was also proposed. In particular, Zn2 was proposed to be tetracoordinated with D86, C164, H206, and a bridging hydroxide ion, with a salt bridge formed between the C4 carboxylate group and a nearby lysine residue (K167). Remarkably, our ampicillin binding model is quite different from the model from Kim et al. However, the strong interactions with K211 and N220 clearly can provide a reasonable explanation for the almost-complete deactivation of the enzyme by the mutation of K211. Based on our simulations on NDM-1 and previous simulations on CcrA, we propose that, for dizinc B1 MβLs, the common substrate binding feature might be that there is no direct contact between the substrate and the Zn2 cofactor. This conclusion is tentative and calls for more studies. On the other F

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Therefore, we cannot exclude the change of the O9−Zn2 distance from the set of reaction coordinates. Therefore, to obtain a complete understanding of the overall processes, we then define a new set of reaction coordinates as follows: d1 = dOw−C7 − dC7−N4 and d2 = dO9−Zn2 − dZn2−Oδ2(D124). 3.3.2. Reaction Path. The calculated two-dimensional PES along the new set of reaction coordinates is displayed in Figure 4A. Corresponding snapshots of all three stationary states are given in Figure 5. Some selected key geometric parameters for

Table 3. Selected Distances (Å) for Stationary Points along the Reaction Path of Ampicillin Hydrolyzed by NDM-1 SCC-DFTB/MM distance Ow···C7 O8··C7 N4···C7 Hw···Oδ1(D124) Ow···Hw Zn1···Zn2 Zn1···O8 Zn1···Ow Zn1···Nε2(H189) Zn1···Nδ1(H122) Zn1···Nε2(H120) Zn2···Nε2(H250) Zn2···Oδ2(D124) Zn2···S(C208) Zn2··· Ow Zn2···N4 Zn2···O9 Hζ1(K211)···O10 Hζ1(K211)···O9 Hζ2(K211)··· O(C208)

DFT

ES complex

TS

EI complex

RC

TS

INT

2.88 1.22 1.41 1.87 0.99 3.52 3.43 2.03 1.99 2.00 2.02 2.00 2.11 2.30 2.02 3.78 4.80 2.57 1.62 1.88

1.95 1.23 1.55 1.67 1.01 3.62 3.18 2.04 1.99 2.01 2.06 2.00 2.43 2.30 2.29 2.41 2.53 1.35 2.58 1.77

1.34 1.25 2.47 1.02 1.87 4.00 2.90 2.08 1.98 2.02 2.00 2.02 3.87 2.31 3.19 1.91 2.16 1.73 2.27 1.77

3.98 1.21 1.39 1.75 0.99 3.58 4.45 1.91 2.03 2.04 2.01 2.07 1.98 2.30 1.98 5.14 5.32 1.60 2.52 1.82

2.02 1.23 1.45 1.62 1.01 3.83 3.28 1.92 2.03 2.06 2.02 2.15 2.11 2.37 2.24 2.49 2.19 1.52 2.64 1.80

1.30 1.24 2.83 1.00 1.77 5.26 2.90 1.97 2.01 2.02 2.03 2.05 3.34 2.33 4.13 1.94 2.10 1.61 2.48 1.80

work, we focus only on the first step of the hydrolysis of ampicillin. Overall, the initial step of the ring-opening reaction in the hydrolysis of ampicillin catalyzed by NDM-1 is a concerted process, in which only one TS can be located to connect the ES and enzyme−intermediate (EI) complexes. The corresponding mechanism is summarized in Scheme 2. To form the EI complex, a barrier of about 10.0 kcal/mol must be overcome. Unlike in L1-catalyzed moxalactam hydrolysis, there is no metastable intermediate related to the proton transfer from the bridging hydroxide ion to D124 in the NDM-1-catalyzed hydrolysis reaction. It is observed that such proton transfer occurs along with the nucleophilic addition by hydroxide ion at the carbonyl carbon (C7) and the cleavage of the amide C7−N4 bond. Notably, the protonation of D124 can expel the aspartic acid residue away from Zn2, as evidenced by the increase in dZn2···Oδ2(D124) from 2.11 Å at the ES complex to 3.87 Å at the EI complex. However, the tetracoordination of Zn2 is still maintained, for which the amide nitrogen anion (N4) and C3 carboxylate group become one of four ligands of the Zn2 cofactor. The cleavage of the C−N bond could lead to the generation of a nitrogen anion, which makes the formation of the N4−Zn2 ligand bond spontaneous. It should also be pointed out that the N4−Zn2 distance at the EI complex was calculated to be 1.91 Å, which is about 0.27 Å shorter than the experimental value of 2.18 Å. Such a deviation does not seem to be a simple computational error, because we observed the same phenomenon in the CphA-catalyzed hydrolysis of the biapenem molecule.18,35 This indicates that the formation of the crystal anionic nitrogen intermediate might occur in the subsequent hydrolysis reaction. Many more simulations are needed to obtain a complete understanding of the overall hydrolysis process. At the same time, the C3 carboxylate group moves down to become the fourth ligand of Zn2, and the C−N bond is cleaved.

Figure 5. Snapshots of stationary states along the putative reaction coordinates in the NDM-1-catalyzed hydrolysis of ampicillin.

all three stationary states are summarized in Table 3. In a recent work, Crowder and co-workers15 suggested that the protonation of the nitrogen anion might be the rate-limiting step. However, it should be noted that they used an N-terminal modified enzyme in their studies. Nevertheless, the lactam ringopening step is still one of the most important steps during the hydrolysis of lactam antibiotics catalyzed by lactamases. In fact, it has also been suggested that the ring-opening step is ratelimiting in other MβLs such as L117,56 and CphA.35,46 In this G

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Scheme 2. Proposed Mechanism for the Initial Lactam Ring Opening of the NDM-1-Catalyzed Hydrolysis of Ampicillin

Figure 6. (Left panel) Two-dimensional potential of mean force for the initial ring-opening step in the hydrolysis of ampicillin catalyzed by NDM-1. (Right panel) Minimum-free-energy curves obtained with the SCC-DFTB/MM model along the reaction coordinate of d1 with (red line) and without (blue line) corrections based on B3LYP/MM calculations.

simply to provide stabilization of the substrate and reactive intermediate during the reaction. Hence, a common functional feature for Zn1 could be proposed among MβLs that might vary depending on the existence of the asparagine residue. If there is an asparagine residue such as N220 in NDM-1 that forms a hydrogen bond with the lactam carbonyl oxygen atom, Zn1 can only provide stabilization for the nucleophile of hydroxide oxygen atom and cannot serve as the oxyanion hole. On the contrary, if there is no such asparagine residue such as L1 and CcrA, Zn1 could play the double role of stabilizing the intermediate and providing the oxyanion hole to polarize the βlactam caronbyl oxygen atom. 3.4. Potential of Mean Force. To account for the entropic effect, we further performed two-dimensional potential of mean force (PMF) simulations along the putative reaction coordinates. The structures obtained by the minimum-energy surface calculations were employed as the initial structures. A total of 805 windows were included in the PMF calculations. For each window, a 100-ps constraint MD simulation was carried out, with the first 60 ps for equilibration and the remaining 40 ps for final PMF generation. Umbrella sampling57 with a harmonic constraint of 150 kcal/(mol·Å2) was applied to each window to enhance the sampling around the transitionstate area. Finally, the two-dimensional weighted histogram analysis method58,59 was employed to obtain the final free energy surface, which is displayed in the left panel of Figure 6. To obtain a better view of the activation energy for the NDM-1-catalyzed hydrolysis of ampicillin, we further plot a minimum-free-energy path along the reaction coordinate (d1) of nucleophilic addition and cleavage of the CN bond in the right panel of Figure 6. As with the reaction path calculations, only one TS could be located to connect the ES and EI

The corresponding distance between O9 and Zn2 is decreased from 4.80 to 1.91 Å. Such a phenomenon suggests that, in the NDM-1-catalyzed hydrolysis of ampicillin, the experimentally observed coordination between the C3 carboxylate group and Zn2 occurs after the initial step of lactam ring opening. This observation is clearly different from the widely accepted lactam antibiotics binding features in other members of the MβL family, as mentioned above, in which the substrate C3/C4 carboxylate group is ligated with Zn2 in the ES complex.5 Although we believe that our model should be reasonable, it is still awaiting more experimental investigations for definitive evidence. The binding feature of Zn1 along the reaction also deserves some discussion. With the bridging hydroxide ion approaching the C7 atom and the cleavage of the C−N bond, a new carboxylate group is generated that is further ligated by Zn1. In contrast to the function of Zn1 in L1 or CcrA, in which it is suggested to serve as the oxyanion hole to stabilize the intermediate,17,48 we could not identify such a contribution provided by Zn1 in NDM-1, as evidenced by the distance between Zn1 and the carbonyl oxygen (O8) being greater than 2.9 Å. A major reason seems to be the substrate binding difference between NDM-1 and L1, for which an asparagineresidue hydrogen bonded with the lactam carbonyl oxygen atom can be observed in NDM-1 but not in L1. Indeed, this hydrogen bond is relatively strong in NDM-1 according to the QM/MM MD simulations as shown above. During the reaction, the hydrogen bond is also well maintained, as shown in Table 3. N220 is thus thought to serve as the oxyanion hole to stabilize the tetrahedral intermediate, which is similar to the function of N233 in the CphA-catalyzed hydrolysis of biapenem. To this point, the function of Zn1 is H

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identified. The mechanism is consistent with our QM/MM simulations. The calculated geometric parameters are given in Table 3, and the energetic profiles are listed in Table 4. The

complexes. The calculated activation energy barrier was about 9.7 kcal/mol, which is significantly smaller than the experimentally estimated value (15.8 kcal/mol) derived from the kinetic analysis (kcat = 15 s−1). The position seems to be at the same position as that obtained in minimum-energy surface calculations. Quite surprisingly, the ring-opening step could release nearly 25 kcal/mol of energy to generate the EI complex. Such a large exothermicity in the SCC-DFTB/MM PMF might be due to some inherent errors in the semiempirical treatment of the QM region. Similar underestimation of the activation energy barrier was recently observed by some other researchers,17 and a possible reason might be attributed to a higher bonding energy in SCC-DFTB than in ab initio or DFT methods.60 To tackle the inaccuracy existing in our PMF calculation, we have further performed single-point calculations at the B3LYP/MM//SCC-DFTB/ MM level of theory. The calculations are carried out using a CHARMM/Gamess-UK interface.61 A standard basis set of 631G(d) was applied to atoms including C, H, O, N, and S elements in the QM region, whereas the LANL2 basis set is used for the zinc atoms with effective core potentials. As shown in Figure 4A, a two-dimensional minimum-energy surface for the initial ring-opening step has been obtained using the SCCDFTB/MM method. First, we plot the minimum-energy curve along the reaction coordinate of d1 in Figure 4B. To reduce the computational cost, B3LYP/MM//SCC-DFTB/MM singlepoint calculations were then carried out for those structures along the minimum-energy path with respect to the reaction coordinate of d1. The energy curve at the B3LYP/MM//SCCDFTB/MM level of theory is also included in Figure 4B. A fourth-order polynomial fitting was applied to obtain the correction of the energy gap between the SCC-DFTB/MM and B3LYP/MM calculations (see more details in the Supporting Information). Finally, such energy corrections were applied to improve the minimum-free-energy path. The corrected free energy curve is finally given in the right panel of Figure 6B. The corresponding activation energy barrier was calculated to be about 19.4 kcal/mol, which is in much better agreement with the experimental value of 15.8 kcal/mol than the original value (9.7 kcal/mol) obtained by the SCC-DFTB/MM method. Moreover, the very large exothermicity was also corrected with this approach. The subsequent processes of hydrolysis are not considered here because the major objective in this work is to clarify a possible enzyme−substrate complex. The following hydrolysis might require additional water molecule which could serve as the origin of the proton to neutralize the nitrogen anion, and generate the new bridging hydroxide ion. Such mechanism has been proposed for the hydrolysis of biapenem catalyzed by CphA.18 On the other hand, in a recent study by Crowder and co-workers15 suggested the formation of the nitrogen anionic intermediate or the protonation of the nitrogen atom should be rate-limiting. However, for the B3 L1 catalyzed hydrolysis of b-lactam compounds, the reaction is limited by the ring-opening partial reaction according Spencer et al.62 Therefore, the comparison of the rate constant obtained by the theoretical and experimental approaches might be just tentative. Additional and more thorough simulations involving subsequent hydrolysis steps are highly required. Efforts in this direction are underway in our laboratory. 3.5. DFT Results for the Truncated Active-Site Model. DFT investigations of the truncated active-site model also revealed that the initial ring-opening of the hydrolysis of ampicillin is a one-step reaction, in which only one TS can be

Table 4. Energetics (kcal/mol) for the Noncatalyzed Hydrolysis Reaction of Ampicillin Using a Truncated ActiveSite Model of NDM-1 Obtained at the B3LYP/6-31G(d) Level of Theory method

RC

TS

INT

B3LYP/6-31G(d) B3LYP/6-31+G(d,p)//B3LYP/6-31G(d) free energy PCM (with ε = 5) PCM (with ε = 80)

0.00 0.00 0.00 0.00 0.00

21.93 21.50 25.47 21.82 21.35

−9.78 −9.53 −8.90 −5.33 −3.52

calculated energy barrier is 21.9 kcal/mol, which is close to our corrected QM/MM free energy barrier height. From Table 4, the inclusion of the diffuse function does not significantly change the energy barrier and the exothermicity, which indicates that the basis set used in the computations is sufficient for further discussions. The free energy barrier was calculated to be 25.5 kcal/mol because of the lack of the contribution of the protein environment. The effect of solvent in the water environment (ε = 80) and the protein environment (ε = 5) examined using PCM model63 does not change the overall energy barrier height, but reduces the exothermicity by several kilocalories per mole to make the overall reaction nearly thermally neutral, which is consistent with our corrected PMF calculations. For the TS, normal-mode frequency analysis indicated only one imaginary frequency, which features a complete concerted motion involving nucleophilic addition, cleavage of the lactam amide bond, formation of a nitrogen anionic intermediate, and formation of O9−Zn2 ligand bond. According to the optimized geometries reported in Table 3 and Figure 7, one can easily see that, with the approach of Ow to C7 (distance decreases from 3.98 Å at the RC to 2.02 Å at the TS), the amide C7−N4 bond is elongated from 1.39 to 1.45 Å. At the same time, the distance between O9 and Zn2 is sharply reduced to 2.19 Å at the TS compared to 5.32 Å at the RC, which clearly supports our QM/ MM simulations that the substrate-metal−ligand bond should be formed after the initial step of ring opening. The proton of the bridging hydroxide ion is simultaneously transferred to D124 to destroy the ligand bond between Zn2 and D124, as evidenced by the fact that the Zn2···Oδ2 distance is elongated to 2.11 Å and finally to 3.34 Å. The formation of the nitrogen anionic intermediate is also supported by the change in the distance between N4 and Zn2 (5.14 Å at the RC vs 1.94 Å at the INT). For the optimized reactant complex, it seems that the substrate has somewhat loose interactions with the amino acid analogues. In particular, the nonbonded distances are much longer than those obtained by the SCC-DFTB/MM method; for example, the distance between Ow and C7 is 3.98 Å compared to 2.88 Å in the SCC-DFTB/MM frame. This difference can be attributed to the lack of protein environment and especially the exclusion of N220 from our truncated model computations. In a word, we stress here that the structure obtained using the QM/MM method is more reliable. The two zinc ions are tetracoordinated. Some interesting structural characteristics should be noted for both RC and INT. In particular, similarly to the QM/MM ES complex, no direct I

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Figure 7. Stationary states of the hydrolysis of ampicillin using the truncated active-site model calculated at the B3LYP/6-31G(d) level of theory.

general substrate binding mode for dizinc B1 MβLs that there might not be a ligand bond formed between the C3/C4 carboxylate group and the metal ion at the second binding position, Zn2. Of course, a such substrate binding proposal requires more computations for confirmation, such as other types of β-lactam-containing substrates. In this work, we also investigated the initial ring-opening step in the hydrolysis of ampicillin based on our simulated Michaelis complex structure using the combined SCC-DFTB/MM method. The reaction shows a completely concerted process involving the nucleophilic addition of the bridging hydroxide at the lactam carbonyl carbon center, the cleavage of the lactam C−N bond, the formation of a nitrogen anionic intermediate, and the formation of a ligand bond between the C3 carboxylate group and Zn2. A subsequent truncated active-site model by DFT computations also confirmed this reaction mechanism. In particular, our simulations demonstrated that the abovementioned O9−Zn2 ligand bond should be formed with the generation of an anionic nitrogen intermediate rather than being formed at the initial substrate binding. The function of Zn1 seems to be highly dependent on the contribution of an active-site asparagine residue. The Zn1 cofactor might serve as the oxyanion hole if there is no asparagine residue that forms a hydrogen bond with the lactam carbonyl oxygen. Because of the presence of N220 in NDM-1, Zn1 cannot play the role of an oxyanion hole but, rather, just provides help in positioning the nucleophile of the hydroxide ion. It should be noted that the subsequent protonation of the nitrogen anion was not investigated here. More calculations are underway in this direction in our laboratory. It is our hope that our simulations will contribute to the ultimate design of clinically useful inhibitors against NDM-1 and other MβLs.

interaction between the C3 carboxylate group and Zn2 was found in the DFT-optimized RC, as confirmed by the IRC calculations. In addition, the cleavage of the C−N bond makes the active site more opening to accommodate additional water molecules for further hydrolysis reaction. To further examine the accuracy of the SCC-DFTB method in describing the hydrolysis of ampicillin, we performed some calculations using the SCC-DFTB method on the same truncated active-site model as used for the DFT investigations. The obtained geometry parameters are reported in Table S1 (Supporting Information), and the corresponding energy profile is displayed in Figure S2 (Supporting Information). For comparison, the results obtained using DFT methods are also included. The calculated barrier height is about 10.7 kcal/ mol, which is close to the free energy barrier height (9.7 kcal/ mol) obtained with the SCC-DFTB/MM model. On the other hand, we also observed a large exothermicity (∼31.8 kcal/mol). Compared to the energetics of the DFT simulations, the overall trend is quite similar to that obtained by B3LYP/MM//SCCDFTB/MM single-point calculations. Clearly, the energy correction for the free energy barrier is necessary. Nevertheless, based on the structural information presented in Table S1 (Supporting Information), one can see that SCC-DFTB and B3LYP/6-31G(d) agree very well with each other. These results further ensure the validity of the SCC-DFTB approach for systems such as zinc-containing enzymes.

4. CONCLUSIONS A newly identified MβL, NDM-1, can hydrolyze nearly all βlactam antibiotics including the so-called “last resort” of carbapenems, thus causing worldwide concern and health threats. The situation could even worsen because of the lack of clinically useful inhibitors. It is then urgent to perform extensive simulations to understand the substrate binding pattern and corresponding catalytic mechanism. In this work, extensive hybrid QM/MM simulations were applied to address the substrate binding pattern for one of the natural substrates of NDM-1, ampicillin. The two zinc ions were found to be tetracoordinated, in which aside from ligands provided by the enzyme, the fourth ligand is a bridging hydroxide ion connecting the two zinc ions. N220 is one of the key residues for stabilizing ampicillin through a hydrogen bond with the C3 carboxylate. It could also play the role of an oxyanion hole to stabilize the hydrolyzed intermediate through a strong hydrogen bond with the lactam carbonyl oxygen atom. Additional stabilization for substrate binding is provided by a nearby lysine residue, K211. Combined with a previous simulation on nitrocefin binding in CcrA48 and the ampicillin binding in NDM-1 obtained in this work, we further propose a

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ASSOCIATED CONTENT

S Supporting Information *

Computational strategies for the correction of the free energy profile for the initial ring-opening step of the hydrolysis of ampicillin catalyzed by NDM-1. Geometric parameters of stationary states for the truncated active-site model optimized using the SCC-DFTB method for comparison. Cartesian coordinates for the truncated active-site model calculated at the B3LYP level of theory. Full citations of refs 19, 27, 30, 43, and 55. 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]. Tel.: 86-28-84752094. J

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Notes

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The authors declare no competing financial interest.

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

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