Ab initio QM/MM study of class A beta-lactamase acylation: dual participation of Glu166 and Lys73 in a concerted base promotion of Ser70

Beta-lactamase acquisition is the most prevalent basis for Gram-negative bacteria resistance to the beta-lactam antibiotics. The mechanism used by the most common class A Gram-negative beta-lactamases is serine acylation followed by hydrolytic deacylation, destroying the beta-lactam. The ab initio quantum mechanical/molecular mechanical (QM/MM) calculations, augmented by extensive molecular dynamics simulations reported herein, describe the serine acylation mechanism for the class A TEM-1 beta-lactamase with penicillanic acid as substrate. Potential energy surfaces (based on approximately 350 MP2/6-31+G calculations) reveal the proton movements that govern Ser70 tetrahedral formation and then collapse to the acyl-enzyme. A remarkable duality of mechanism for tetrahedral formation is implicated. Following substrate binding, the pathway initiates by a low energy barrier (5 kcal mol(-1)) and an energetically favorable transfer of a proton from Lys73 to Glu166, through the catalytic water molecule and Ser70. This gives unprotonated Lys73 and protonated Glu166. Tetrahedral formation ensues in a concerted general base process, with Lys73 promoting Ser70 addition to the beta-lactam carbonyl. Moreover, the three-dimensional potential energy surface also shows that the previously proposed pathway, involving Glu166 as the general base promoting Ser70 through a conserved water molecule, exists in competition with the Lys73 process. The existence of two routes to the tetrahedral species is fully consistent with experimental data for mutant variants of the TEM beta-lactamase.     

The deacylation mechanism of AmpC beta-lactamase at ultrahigh resolution

Beta-lactamases confer bacterial resistance to beta-lactam antibiotics, such as penicillins. The characteristic class C beta-lactamase AmpC catalyzes the reaction with several key residues including Ser64, Tyr150, and Lys67. Here, we describe a 1.07 A X-ray crystallographic structure of AmpC beta-lactamase in complex with a boronic acid deacylation transition-state analogue. The high quality of the electron density map allows the determination of many proton positions. The proton on the Tyr150 hydroxyl group is clearly visible and is donated to the boronic oxygen mimicking the deacylation water. Meanwhile, Lys67 hydrogen bonds with Ser64Ogamma, Asn152Odelta1, and the backbone oxygen of Ala220. This suggests that this residue is positively charged and has relinquished the hydrogen bond with Tyr150 observed in acyl-enzyme complex structures. Together with previous biochemical and NMR studies, these observations indicate that Tyr150 is protonated throughout the reaction coordinate, disfavoring mechanisms that involve a stable tyrosinate as the general base for deacylation. Rather, the hydroxyl of Tyr150 appears to be well positioned to electrostatically stabilize the negative charge buildup in the tetrahedral high-energy intermediate. This structure, in itself, appears consistent with a mechanism involving either Tyr150 acting as a transient catalytic base in conjunction with a neutral Lys67 or the lactam nitrogen as the general base. Whereas mutagenesis studies suggest that Lys67 may be replaced by an arginine, disfavoring the conjugate base mechanism, distinguishing between these two hypotheses may ultimately depend on direct determination of the pK(a) of Lys67 along the reaction coordinate.     

Mechanisms of antibiotic resistance: QM/MM modeling of the acylation reaction of a class A beta-lactamase with benzylpenicillin

Understanding the mechanisms by which beta-lactamases destroy beta-lactam antibiotics is potentially vital in developing effective therapies to overcome bacterial antibiotic resistance. Class A beta-lactamases are the most important and common type of these enzymes. A key process in the reaction mechanism of class A beta-lactamases is the acylation of the active site serine by the antibiotic. We have modeled the complete mechanism of acylation with benzylpenicillin, using a combined quantum mechanical and molecular mechanical (QM/MM) method (B3LYP/6-31G+(d)//AM1-CHARMM22). All active site residues directly involved in the reaction, and the substrate, were treated at the QM level, with reaction energies calculated at the hybrid density functional (B3LYP/6-31+Gd) level. Structures and interactions with the protein were modeled by the AM1-CHARMM22 QM/MM approach. Alternative reaction coordinates and mechanisms have been tested by calculating a number of potential energy surfaces for each step of the acylation mechanism. The results support a mechanism in which Glu166 acts as the general base. Glu166 deprotonates an intervening conserved water molecule, which in turn activates Ser70 for nucleophilic attack on the antibiotic. This formation of the tetrahedral intermediate is calculated to have the highest barrier of the chemical steps in acylation. Subsequently, the acylenzyme is formed with Ser130 as the proton donor to the antibiotic thiazolidine ring, and Lys73 as a proton shuttle residue. The presented mechanism is both structurally and energetically consistent with experimental data. The QM/MM energy barrier (B3LYP/ 6-31G+(d)//AM1-CHARMM22) for the enzymatic reaction of 9 kcal mol(-1) is consistent with the experimental activation energy of about 12 kcal mol(-1). The effects of essential catalytic residues have been investigated by decomposition analysis. The results demonstrate the importance of the "oxyanion hole" in stabilizing the transition state and the tetrahedral intermediate. In addition, Asn132 and a number of charged residues in the active site have been identified as being central to the stabilizing effect of the enzyme. These results will be potentially useful in the development of stable beta-lactam antibiotics and for the design of new inhibitors.     

Insights into the acylation mechanism of class A beta-lactamases from molecular dynamics simulations of the TEM-1 enzyme complexed with benzylpenicillin

Herein, we present results from molecular dynamics MD simulations ( approximately 1 ns) of the TEM-1 beta-lactamase in aqueous solution. Both the free form of the enzyme and its complex with benzylpenicillin were studied. During the simulation of the free enzyme, the conformation of the Omega loop and the interresidue contacts defining the complex H-bond network in the active site were quite stable. Most interestingly, the water molecule connecting Glu166 and Ser70 does not exchange with bulk solvent, emphasizing its structural and catalytic relevance. In the presence of the substrate, Ser130, Ser235, and Arg244 directly interact with the beta-lactam carboxylate via H-bonds, whereas the Lys234 ammonium group has only an electrostatic influence. These interactions together with other specific contacts result in a very short distance ( approximately 3 A) between the attacking hydroxyl group of Ser70 and the beta-lactam ring carbonyl group, which is a favorable orientation for nucleophilic attack. Our simulations also gave insight into the possible pathways for proton abstraction from the Ser70 hydroxyl group. We propose that either the Glu166 carboxylate-Wat1 or the substrate carboxylate-Ser130 moieties could abstract a proton from the nucleophilic Ser70.     

Investigation of the mechanism of the cell wall DD-carboxypeptidase reaction of penicillin-binding protein 5 of Escherichia coli by quantum mechanics/molecular mechanics calculations

Penicillin-binding protein 5 (PBP 5) of Escherichia coli hydrolyzes the terminal D-Ala-D-Ala peptide bond of the stem peptides of the cell wall peptidoglycan. The mechanism of PBP 5 catalysis of amide bond hydrolysis is initial acylation of an active site serine by the peptide substrate, followed by hydrolytic deacylation of this acyl-enzyme intermediate to complete the turnover. The microscopic events of both the acylation and deacylation half-reactions have not been studied. This absence is addressed here by the use of explicit-solvent molecular dynamics simulations and ONIOM quantum mechanics/molecular mechanics (QM/MM) calculations. The potential-energy surface for the acylation reaction, based on MP2/6-31+G(d) calculations, reveals that Lys47 acts as the general base for proton abstraction from Ser44 in the serine acylation step. A discrete potential-energy minimum for the tetrahedral species is not found. The absence of such a minimum implies a conformational change in the transition state, concomitant with serine addition to the amide carbonyl, so as to enable the nitrogen atom of the scissile bond to accept the proton that is necessary for progression to the acyl-enzyme intermediate. Molecular dynamics simulations indicate that transiently protonated Lys47 is the proton donor in tetrahedral intermediate collapse to the acyl-enzyme species. Two pathways for this proton transfer are observed. One is the direct migration of a proton from Lys47. The second pathway is proton transfer via an intermediary water molecule. Although the energy barriers for the two pathways are similar, more conformers sample the latter pathway. The same water molecule that mediates the Lys47 proton transfer to the nitrogen of the departing D-Ala is well positioned, with respect to the Lys47 amine, to act as the hydrolytic water in the deacylation step. Deacylation occurs with the formation of a tetrahedral intermediate over a 24 kcal x mol(-1) barrier. This barrier is approximately 2 kcal x mol(-1) greater than the barrier (22 kcal x mol(-1)) for the formation of the tetrahedral species in acylation. The potential-energy surface for the collapse of the deacylation tetrahedral species gives a 24 kcal x mol(-1) higher energy species for the product, signifying that the complex would readily reorganize and pave the way for the expulsion of the product of the reaction from the active site and the regeneration of the catalyst. These computational data dovetail with the knowledge on the reaction from experimental approaches.     

Inhibition of class A beta-lactamases by carbapenems: crystallographic observation of two conformations of meropenem in SHV-1

Carbapenem antibiotics are often the "last resort" in the treatment of infections caused by bacteria resistant to penicillins and cephalosporins. To understand why meropenem is resistant to hydrolysis by the SHV-1 class A beta-lactamase, the atomic structure of meropenem inactivated SHV-1 was solved to 1.05 A resolution. Two conformations of the Ser70 acylated intermediate are observed in the SHV-1-meropenem complex; the meropenem carbonyl oxygen atom of the acyl-enzyme is in the oxyanion hole in one conformation, while in the other conformation it is not. Although the structures of the SHV-1 apoenzyme and the SHV-1-meropenem complex are very similar (0.29 A rmsd for Calpha atoms), the orientation of the conserved Ser130 is different. Notably, the Ser130-OH group of the SHV-1-meropenem complex is directed toward Lys234Nz, while the Ser130-OH of the apo enzyme is oriented toward the Lys73 amino group. This altered position may affect proton transfer via Ser130 and the rate of hydrolysis. A most intriguing finding is the crystallographic detection of protonation of the Glu166 known to be involved in the deacylation mechanism. The critical deacylation water molecule has an additional hydrogen-bonding interaction with the OH group of meropenem's 6alpha-1 R-hydroxyethyl substituent. This interaction may weaken the nucleophilicity and/or change the direction of the lone pair of electrons of the water molecule and result in poor turnover of meropenem by the SHV-1 beta-lactamase. Using timed mass spectrometry, we further show that meropenem is covalently attached to SHV-1 beta-lactamase for at least 60 min. These observations explain key properties of meropenem's ability to resist hydrolysis by SHV-1 and lead to important insights regarding future carbapenem and beta-lactamase inhibitor design.     

A theoretical study on the substrate deacylation mechanism of class C beta-lactamase

The whole reaction of the deacylation of class C beta-lactamase was investigated by performing quantum chemical calculations under physiological conditions. In this study, the X-ray crystallographic structure of the inhibitor moxalactam-bound class C beta-lactamase (Patera et al. J. Am. Chem. Soc. 2000, 122, 10504-10512.) was utilized and moxalactam was changed into the substrate cefaclor. A model for quantum chemical calculations was constructed using an energy-minimized structure of the substrate-bound enzyme obtained by molecular mechanics calculation, in which the enzyme was soaked in thousands of TIP3P water molecules. It was found that the deacylation reaction consisted of two elementary processes. The first process was formation of a tetrahedral intermediate, which was initiated by the activation of catalytic water by Tyr150, and the second process was detachment of the hydroxylated substrate from the enzyme, which associated with proton transfer from the side chain of Lys67 to Ser64O(gamma). The first process is a rate-determining process, and the activation energy was estimated to be 30.47 kcal/mol from density functional theory calculations considering electron correlation (B3LYP/6-31G**). The side chain of Tyr150 was initially in a deprotonated state and was stably present in the active site of the acyl-enzyme complex, being held by Lys67 and Lys315 cooperatively.     

pKa,MM, and QM studies of mechanisms of beta-lactamases and penicillin-binding proteins: acylation step

The acylation step of the catalytic mechanism of beta-lactamases and penicillin-binding proteins (PBPs) has been studied with various approaches. The methods applied range from molecular dynamics (MD) simulations to multiple titration calculations using the Poisson-Boltzmann approach to quantum mechanical (QM) methods. The mechanism of class A beta-lactamases was investigated in the greatest detail. Most approaches support the critical role of Glu-166 and hydrolytic water in the acylation step of the enzymatic catalysis in class A beta-lactamases. The details of the catalytic mechanism have been revealed by the QM approach, which clearly pointed out the critical role of Glu-166 acting as a general base in the acylation step with preferred substrates. Lys-73 shuffles a proton abstracted by Glu-166 O(epsilon ) to the beta-lactam nitrogen through Ser-130 hydroxyl. This proton is transferred from O(gamma) of the catalytic Ser-70 through the bridging hydrolytic water to Glu-166 O(epsilon ). Then the hydrogen is simultaneously passed through S(N)2 inversion mechanism at Lys-73 N(zeta) to Ser-130 O(gamma), which loses its proton to the beta-lactam nitrogen. The protonation of beta-lactam nitrogen proceeds with an immediate ring opening and collapse of the first tetrahedral species into an acyl-enzyme intermediate. However, the studies that considered the effect of solvation lower the barrier for the pathway, which utilizes Lys-73 as a general base, thus creating a possibility of multiple mechanisms for the acylation step in the class A beta-lactamases. These findings help explain the exceptional efficiency of these enzymes. They emphasize an important role of Glu-166, Lys-73, and Ser-130 for enzymatic catalysis and shed light on details of the acylation step of class A beta-lactamase mechanism. The acylation step for class C beta-lactamases and six classes of PBPs were also considered with continuum solvent models and MD simulations.     

Role of protein flexibility in enzymatic catalysis: quantum mechanical-molecular mechanical study of the deacylation reaction in class A beta-lactamases

We present a theoretical study of a mechanism for the hydrolysis of the acyl-enzyme complex formed by a class A beta-lactamase (TEM1) and an antibiotic (penicillanate), as a part of the process of antibiotic's inactivation by this type of enzymes. In the presented mechanism the carboxylate group of a particular residue (Glu166) activates a water molecule, accepting one of its protons, and afterward transfers this proton directly to the acylated serine residue (Ser70). In our study we employed a quantum mechanics (AM1)-molecular mechanics partition scheme (QM/MM) where all the atoms of the system were allowed to relax. For this purpose we used the GRACE procedure in which part of the system is used to define the Hessian matrix while the rest is relaxed at each step of the stationary structures search. By use of this computational scheme, the hydrolysis of the acyl-enzyme is described as a three-step process: The first step corresponds to the proton transfer from the hydrolytic water molecule to the carboxylate group of Glu166 and the subsequent formation of a tetrahedral adduct as a consequence of the attack of this activated water molecule to the carbonyl carbon atom of the beta-lactam. In the second step, the acyl-enzyme bond is broken, obtaining a negatively charged Ser70. In the last step this residue is protonated by means of a direct proton transfer from Glu166. The large mobility of Glu166, a residue that is placed in a Ohms-loop, is essential to facilitate this mechanism. The geometry of the acyl-enzyme complex shows a large distance between Glu166 and Ser70 and thus, if protein coordinates were kept frozen during the reaction path, it would be difficult to get a direct proton transfer between these two residues. This computational study shows how a flexible treatment suggests the feasibility of a mechanism that could have been discounted on the basis of crystallographic positions.     

Ab initio model study on acetylcholinesterase catalysis: potential energy surfaces of the proton transfer reactions

Ab initio molecular orbital (MO) and hybrid density functional theory (DFT) calculations have been applied to the initial step of the acylation reaction catalyzed by acetylcholinesterase (AChE), which is the nucleophiric addition of Ser200 in catalytic triads to a neurotransmitter acetylcholine (ACh). We focus our attention mainly on the effects of oxyanion hole and Glu327 on the potential energy surfaces (PESs) for the proton transfer reactions in the catalytic triad Ser200-His440-Glu327. The activation barrier for the addition reaction of Ser200 to ACh was calculated to be 23.4 kcal/mol at the B3LYP/6-31G(d)//HF/3-21G(d) level of theory. The barrier height under the existence of oxyanion hole, namely, Ser200-His440-Glu327-ACh-(oxyanion hole) system, decreased significantly to 14.2 kcal/mol, which is in reasonable agreement with recent experimental value (12.0 kcal/mol). Removal of Glu327 from the catalytic triad caused destabilization of both energy of transition state for the reaction and tetrahedral intermediate (product). PESs calculated for the proton transfer reactions showed that the first proton transfer process is the most important in the stabilization of tetrahedral intermediate complex. The mechanism of addition reaction of ACh was discussed on the basis of theoretical results.     

Molecular dynamics simulations of the catalytic pathway of a cysteine protease: a combined QM/MM study of human cathepsin K

Molecular dynamics simulations using a combined QM/MM potential have been performed to study the catalytic mechanism of human cathepsin K, a member of the papain family of cysteine proteases. We have determined the two-dimensional free energy surfaces of both acylation and deacylation steps to characterize the reaction mechanism. These free energy profiles show that the acylation step is rate limiting with a barrier height of 19.8 kcal/mol in human cathepsin K and of 29.3 kcal/mol in aqueous solution. The free energy of activation for the deacylation step is 16.7 kcal/mol in cathepsin K and 17.8 kcal/mol in aqueous solution. The reduction of free energy barrier is achieved by stabilization of the oxyanion in the transition state. Interestingly, although the "oxyanion hole" has been formed in the Michaelis complex, the amide units do not donate hydrogen bonds directly to the carbonyl oxygen of the substrate, but they stabilize the thiolate anion nucleophile. Hydrogen-bonding interactions are induced as the substrate amide group approaches the nucleophile, moving more than 2 A and placing the oxyanion in contact with Gln19 and the backbone amide of Cys25. The hydrolysis of peptide substrate shares a common mechanism both for the catalyzed reaction in human cathepsin K and for the uncatalyzed reaction in water. Overall, the nucleophilic attack by Cys25 thiolate and the proton-transfer reaction from His162 to the amide nitrogen are highly coupled, whereas a tetrahedral intermediate is formed along the nucleophilic reaction pathway.     

Ab initio and density functional theory studies of the catalytic mechanism for ester hydrolysis in serine hydrolases

We present results from ab initio and density functional theory studies of the mechanism for serine hydrolase catalyzed ester hydrolysis. A model system containing both the catalytic triad and the oxyanion hole was studied. The catalytic triad was represented by formate anion, imidazole, and methanol. The oxyanion hole was represented by two water molecules. Methyl formate was used as the substrate. In the acylation step, our computations show that the cooperation of the Asp group and oxyanion hydrogen bonds is capable of lowering the activation barrier by about 15 kcal/mol. The transition state leading to the first tetrahedral intermediate in the acylation step is rate limiting with an activation barrier (ΔE₀) of 13.4 kcal/mol. The activation barrier in the deacylation step is smaller. The double-proton-transfer mechanism is energetically unfavorable by about 2 kcal/mol. The bonds between the Asp group and the His group, and the hydrogen bonds in the oxyanion hole, increase in strength going from the Michaelis complex toward the transition state and the tetrahedral intermediate. In the acylation step, the tetrahedral intermediate is a very shallow minimum on the energy surface and is not viable when molecular vibrations are included. © 1998 John Wiley & Sons, Inc. Int J Quant Chem 69: 89–103, 1998     

Role of the catalytic triad and oxyanion hole in acetylcholinesterase catalysis: an ab initio QM/MM study

The initial step of the acylation reaction catalyzed by acetylcholinesterase (AChE) has been studied by a combined ab initio quantum mechanical/molecular mechanical (QM/MM) approach. The reaction proceeds through the nucleophilic addition of the Ser203 O to the carbonyl C of acetylcholine, and the reaction is facilitated by simultaneous proton transfer from Ser203 to His447. The calculated potential energy barrier at the MP2(6-31+G) QM/MM level is 10.5 kcal/mol, consistent with the experimental reaction rate. The third residue of the catalytic triad, Glu334, is found to be essential in stabilizing the transition state through electrostatic interactions. The oxyanion hole, formed by peptidic NH groups from Gly121, Gly122, and Ala204, is also found to play an important role in catalysis. Our calculations indicate that, in the AChE-ACh Michaelis complex, only two hydrogen bonds are formed between the carbonyl oxygen of ACh and the peptidic NH groups of Gly121 and Gly122. As the reaction proceeds, the distance between the carbonyl oxygen of ACh and NH group of Ala204 becomes smaller, and the third hydrogen bond is formed both in the transition state and in the tetrahedral intermediate.     

Structure, function, and inhibition along the reaction coordinate of CTX-M beta-lactamases

CTX-M enzymes are an emerging group of extended spectrum beta-lactamases (ESBLs) that hydrolyze not only the penicillins but also the first-, second-, and third-generation cephalosporins. Although they have become the most frequently observed ESBLs in certain areas, there are few effective inhibitors and relatively little is known about their detailed mechanism. Here we describe the X-ray crystal structures of CTX-M enzymes in complex with different transition-state analogues and beta-lactam inhibitors, representing the enzyme as it progresses from its acylation transition state to its acyl enzyme complex to the deacylation transition state. As the enzyme moves along this reaction coordinate, two key catalytic residues, Lys73 and Glu166, change conformations, tracking the state of the reaction. Unexpectedly, the acyl enzyme complex with the beta-lactam inhibitor cefoxitin still has the catalytic water bound; this water had been predicted to be displaced by the unusual 7alpha-methoxy of the inhibitor. Instead, the 7alpha-group appears to inhibit by preventing the formation of the deacylation transition state through steric hindrance. From an inhibitor design standpoint, we note that the best of the reversible inhibitors, a ceftazidime-like boronic acid compound, binds to CTX-M-16 with a K(i) value of 4 nM. When used together in cell culture, this inhibitor reversed cefotaxime resistance in CTX-M-producing bacteria. The structure of its complex with CTX-M enzyme and the structural view of the reaction coordinate described here provide templates for inhibitor design and intervention to combat this family of antibiotic resistance enzymes.     

Theoretical studies on the deacylation step of serine protease catalysis in the gas phase, in solution, and in elastase

The deacylation step of serine protease catalysis is studied using DFT and ab initio QM/MM calculations combined with MD/umbrella sampling calculations. Free energies of the entire reaction are calculated in the gas phase, in a continuum solvent, and in the enzyme elastase. The calculations show that a concerted mechanism in the gas phase is replaced by a stepwise mechanism when solvent effects or an acetate ion are added to the reference system, with the tetrahedral intermediate being a shallow minimum on the free energy surface. In the enzyme, the tetrahedral intermediate is a relatively stable species ( approximately 7 kcal/mol lower in energy than the transition state), mainly due to the electrostatic effects of the oxyanion hole and Asp102. It is formed in the first step of the reaction, as a result of a proton transfer from the nucleophilic water to His57 and of an attack of the remaining hydroxyl on the ester carbonyl. This is the rate-determining step of the reaction, which requires approximately 22 kcal/mol for activation, approximately 5 kcal/mol less than the reference reaction in water. In the second stage of the reaction, only small energy barriers are detected to facilitate the proton transfer from His57 to Ser195 and the breakdown of the tetrahedral intermediate. Those are attributed mainly to a movement of Ser195 and to a rotation of the His57 side chain. During the rotation, the imidazolium ion is stabilized by a strong H-bond with Asp102, and the C(epsilon)(1)-H...O H-bond with Ser214 is replaced by one with Thr213, suggesting that a "ring-flip mechanism" is not necessary as a driving force for the reaction. The movements of His57 and Ser195 are highly correlated with rearrangements of the binding site, suggesting that product release may be implicated in the deacylation process.     

Phosphorylation reaction in cAPK protein kinase-free energy quantum mechanical/molecular mechanics simulations

We present results of a theoretical analysis of the phosphorylation reaction in cAMP-dependent protein kinase using a combined quantum mechanical and molecular mechanics (QM/MM) approach. Detailed analysis of the reaction pathway is provided using a novel QM/MM implementation of the nudged elastic band method, finite temperature fluctuations of the protein environment are taken into account using free energy calculations, and an analysis of hydrogen bond interactions is performed on the basis of calculated frequency shifts. The late transfer of the substrate proton to the conserved aspartate (D166), the activation free energy of 15 kcal/mol, and the slight exothermic (-3 kcal/mol) character of the reaction are all consistent with the experimental data. The near attack conformation of D166 in the reactant state is maintained by interactions with threonine-201, asparagine-177, and most notably by a conserved water molecule serving as a strong structural link between the primary metal ion and the D166. The secondary Mg ion acts as a Lewis acid, attacking the beta-gamma bridging oxygen of ATP. This interaction, along with a strong hydrogen bond between the D166 and the substrate, contributes to the stabilization of the transition state. Lys-168 maintains a hydrogen bond to a transferring phosphoryl group throughout a reaction process. This interaction increases in the product state and contributes to its stabilization.     

An Unsual Cys-Glu-Lys Catalytic Triad is Responsible for the Catalytic Mechanism of the Nitrilase Superfamily: A QM/MM Study on Nit2

Nitrilase 2 (Nit2) is a representative member of the nitrilase superfamily that catalyzes the hydrolysis of α-ketosuccinamate into oxaloacetate. It has been associated with the metabolism of rapidly dividing cells like cancer cells. The catalytic mechanism of Nit2 employs a catalytic triad formed by Cys191, Glu81 and Lys150. The Cys191 and Glu81 play an active role during the catalytic process while the Lys150 is shown to play only a secondary role. The results demonstrate that the catalytic mechanism of Nit2 involves four steps. The nucleophilic attack of Cys191 to the α-ketosuccinamate, the formation of two tetrahedral enzyme adducts and the hydrolysis of a thioacyl-enzyme intermediate, from which results the formation of oxaloacetate and enzymatic turnover. The rate limiting step of the catalytic process is the formation of the first tetrahedral intermediate with a calculated activation free energy of 18.4 kcal/mol, which agrees very well with the experimental k_cat (17.67 kcal/mol).     

Molecular mechanisms of antibiotic resistance: QM/MM modelling of deacylation in a class A beta-lactamase

Modelling of the first step of the deacylation reaction of benzylpenicillin in the E. coli TEM1 beta-lactamase (with B3LYP/6-31G + (d)//AM1-CHARMM22 quantum mechanics/molecular mechanics methods) shows that a mechanism in which Glu166 acts as the base to deprotonate a conserved water molecule is both energetically and structurally consistent with experimental data; the results may assist the design of new antibiotics and beta-lactamase inhibitors.     

Homology modeling and molecular dynamic simulation of UDP-N-acetylmuramoyl-l-alanine-d-glutamate ligase (MurD) from Mycobacterium tuberculosis H37Rv using in silico approach

The present study aimed to identify the prospective inhibitors of MurD, a cytoplasmic enzyme that catalyzes the addition of d-glutamate to the UDP-N-acetylmuramoyl-l-alanine nucleotide precursor in Mycobacterium tuberculosis (MTB), using virtual screening, docking studies, pharmacokinetic analysis, Molecular Dynamic (MD) simulation, and Molecular Mechanics Generalized Born and Surface Area (MM-GBSA) analyses. The three dimensional (3D) structure was determined based on the homology technique using a template from Streptococcus agalactiae. The modeled structure had three binding sites, namely; substrate binding site (Val18, Thr19, Asp39, Asp40, Gly75, Asn147, Gln171 and His192), the ATP binding site (Gly123, Lys124, Thr125, Thr126, Glu166, Asp283, and Arg314) and the glutamic acid binding site (Arg382, Ser463, and Tyr470). These residues mentioned above play a critical role in the catalytic activity of the enzyme, and their inhibition could serve as a stumbling block to the normal function of the enzyme. A total of 10,344 obtained from virtual screened of Zinc and PubChem databases. These compounds further screened for Lipinski rule of five, docking studies and pharmacokinetic analysis. Four compounds with good binding energies (ZINC11881196 = −10.33 kcal/mol, ZINC12247644 = −8.90 kcal/mol, ZINC14995379 =−8.42 kcal/mol, and PubChem6185 = −8.20 kcal/mol), better than the binding energies of the ATP (−2.31 kcal/mol) and the ligand with known IC₅₀, Aminothiazole (−7.11 kcal/mol) were selected for the MD simulation and MM-GBSA analyses. The result of the analyses showed that all the four ligands formed a stable complex and had the binding free energies better than the binding energy of ATP. Therefore, these ligands considered as suitable prospective inhibitors of the MurD after experimental validation.