A QM/MM study of the catalytic mechanism of aspartate ammonia lyase

Aspartate ammonia lyase (Asp) is one of three types of ammonia lyases specific for aspartate or its derivatives as substrates, which catalyzes the reversible reaction of l-aspartate to yield fumarate and ammonia. In this paper, the catalytic mechanism of Asp has been studied by using combined quantum-mechanical/molecular-mechanical (QM/MM) approach. The calculation results indicate that the overall reaction only contains two elementary steps. The first step is the abstraction of C_β proton of l-aspartate by Ser318, which is calculated to be rate limiting. The second step is the cleavage of C_αN bond of l-aspartate to form fumarate and ammonia. Ser318 functions as the catalytic base, whereas His188 is a dispensable residue, but its protonation state can influence the active site structure and the existing form of leaving amino group, thereby influences the activity of the enzyme, which can well explain the pH dependence of enzymatic activity. Mutation of His188 to Ala only changes the active site structure and slightly elongates the distance of C_β proton of substrate with Ser318, causing the enzyme to remain significant but reduced activity.     

Discerning the catalytic mechanism of Staphylococcus aureus sortase A with QM/MM free energy calculations

Sortases are key virulence factors in Gram-positive bacteria. These enzymes embed surface proteins in the cell wall through a transpeptidation reaction that involves recognizing a penta-peptide “sorting signal” in a target protein, cleaving it, and covalently attaching it to a second substrate that is later inserted into the cell wall. Although well studied, several aspects of the mechanism by which sortases perform these functions remains unclear. In particular, experiments have revealed two potential sorting signal binding motifs: a “Threonine-Out” (Thr-Out) structure in which the catalytically critical threonine residues protrudes into solution, and a “Threonine-In” (Thr-In) configuration in which this residue inserts into the binding site. To determine which of these is the biologically relevant state, we have performed a series of conventional and hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations of the Staphylococcus aureus sortase A (SrtA) enzyme bound to a sorting signal substrate. Through the use of multi-dimensional metadynamics, our simulations were able to both map the acylation mechanism of SrtA in the Thr-In and Thr-Out states, as well as determine the free energy minima and barriers along these reactions. Results indicate that in both states the catalytic mechanisms are similar, however the free energy barriers are lower in the Thr-In configuration, suggesting that Thr-In is the catalytically relevant state. This has important implications for advancing our understanding of the mechanisms of sortase enzymes, as well we for future structure based drug design efforts aimed at inhibiting sortase function in vivo.     

QM/MM modeling of the hydrolysis and transfructosylation reactions of fructosyltransferase from Aspergillus japonicas,an enzyme that produces prebiotic fructooligosaccharide

Fructosyltransferases (FTs) act on sucrose by cleaving the β-(2 → 1) linkage, releasing glucose, and then transferring the fructosyl group to an acceptor molecule. These enzymes are capable of producing prebiotic fructooligosaccharides (FOSs) that are of industrial interest. While several FOS-synthesizing enzymes FTs have been investigated, their catalytic mechanism is not yet fully understood, especially the molecular details of how FOS are enzymatically synthesized from sucrose. Here, we present a comparative quantum mechanics/molecular mechanics (QM/MM) study on the hydrolysis and transfructosylation reactions catalyzed by A. japonicus FT using sucrose as donor and acceptor substrates. It is shown that the hydrolysis and transfructosylation reactions of the enzyme seem to be competitive with similar potential energy profiles. For all studied reaction steps, the fructosyl ring bound in the −1 position was observed to have a ⁴E conformation in the oxocarbonium ion-like transition state. Based on the SCC-DFTB/MM simulations of sucrose complexes of wildtype and D191A mutant FT, Asp191 is shown to be responsible for the productive sugar conformation (at subsite −1) required for catalysis. A key interaction, Asp119⋯nucleophile⋯1–OH (substrate), is proposed to facilitate the formation of fructosyl-enzyme intermediate. This is the first computational study for understanding the FOS synthesis process, and it can be applicable to related FOS-synthesizing enzymes.     

Mechanistic insights into the catalytic reaction of plant allene oxide synthase (pAOS) via QM and QM/MM calculations

QM cluster and QM/MM protein models have been employed to understand aspects of the reaction mechanism of plant allene oxide synthase (pAOS). In this study we have investigated two reaction mechanisms for pAOS. The standard pAOS mechanism was contrasted with an alternative involving an additional active site molecule which has been shown to facilitate proton coupled electron transfer (PCET) in related systems. Firstly, we found that the results from QM/MM protein model are comparable with those from the QM cluster model, presumably due to the large active site used. Furthermore, the results from the QM cluster model show that the Fe^III and Fe^IV pathways for the standard mechanism have similar energetic and structural properties, indicating that the reaction mechanism may well proceed via both pathways. However, while the PCET process is facilitated by an additional active site bound water in other related families, in pAOS it is not, suggesting this type of process is not general to all closely related family members.     

Mechanisms of histone lysine-modifying enzymes: A computational perspective on the role of the protein environment

Epigenetic pathways are involved in a wide range of diseases, including cancer and neurological disorders. Specifically, histone modifying and reading processes are the most broadly studied and are targeted by several licensed drugs. Although there have been significant advances in understanding the mechanistic aspects underlying epigenetic regulation, the development of selective small-molecule inhibitors remains a challenge.Experimentally, it is generally difficult to elucidate the atomistic basis for substrate recognition, as well as the sequence of events involved in binding and the subsequent chemical processes. In this regard, computational modelling is particularly valuable, since it can provide structural features (including transition state structures along with kinetic and thermodynamic parameters) that enable both qualitative and quantitative evaluation of the mechanistic details involved. Here, we summarize knowledge gained from computational modelling studies elucidating the role of the protein environment in histone-lysine modifying and reading mechanisms. We give a perspective on the importance of calculations to aid and advance the understanding of these processes and for the future development of selective inhibitors for epigenetic regulators.     

Novel binding patterns between ganoderic acids and neuraminidase: Insights from docking,molecular dynamics and MM/PBSA studies

Recently, ganoderic acids (GAs) give rise to the attractive candidates of novel neuraminidase (NA) inhibitors. However, there is still no evident conclusion about their binding patterns. To this end, docking, molecular dynamics and MM/PBSA methods were combined to study the binding profiles of GAs with the N1 protein and familiar H274Y and N294S mutations (A/Vietnam/1203/04 stain). It was found that the binding affinities of ganoderic acid DM and Z (ΔG_bind, −16.83 and −10.99 kcal mol⁻¹) are comparable to that of current commercial drug oseltamivir (−23.62 kcal mol⁻¹). Electrostatic interaction is the main driving force, and should be one important factor to evaluate the binding quality and rational design of NA inhibitors. The 150-loop residues Asp151 and Arg152 played an important role in the binding processes. Further analysis revealed that ganoderic acid DM is a potential source of anti-influenza ingredient, with novel binding pattern and advantage over oseltamivir. It had steric hindrance on the 150 cavity of N1 protein, and exerted activities across the H274Y and N294S mutations. This work also pointed out how to effectively design dual-site NA inhibitors and reinforce their affinities. These findings should prove valuable for the in-depth understanding of interactions between NA and GAs, and warrant the experimental aspects to design novel anti-influenza drugs.     

Probing the binding site characteristics of HSA: A combined molecular dynamics and cheminformatics investigation

Human serum albumin is a remarkable protein found in high concentrations in the body. It contains at least seven distinct fatty acid binding sites and two principle sites for drugs. Its primary function is to act as a fatty acid transport system, but it also shows the capacity to bind a diverse range of acidic, neutral and zwitterionic drug molecules. In this paper we investigate the ligand binding selectivity of HSA using cheminformatics analyses and molecular dynamics simulations. We compare and contrast the known ligand binding specificities as obtained from X-ray structural data using PCA, with additional direct analyses of the seven key binding pockets using analyses derived from molecular simulations. We assess both the fatted and defatted states of HSA using 100 ns simulations of the APO and HOLO forms, as well as structures containing one, three and seven myristic acid molecules. We find that differences in fatty acid binding can have a dramatic effect on the flexibility of the protein and also the pocket characteristics. We discuss how the remarkable selectivity of the HSA pockets towards both endogenous fatty acids and exogenous drug molecules is not simply controlled by bulk property effects such as ionization state and lipophilicity.     

MD and QM/MM study on catalytic mechanism of a FAD-dependent enzyme ORF36: For nitro sugar biosynthesis

The catalytic mechanism of a FAD-dependent nitrososynthase (ORF36) was studied with molecular dynamics (MD) and quantum mechanical/molecular mechanical (QM/MM) methods. Residues Leu160 and Phe374 play an important role during the FAD binding with ORF36. Similar phenylalanine/leucine pair was found in the other two enzymes of this family. For the second oxidation step of ORF36 toward thymidine diphosphate-l-epi-vancosamine, three elementary catalytic steps were found: a hydroxylation step, a hydrogen back-transfer step and a hydroxyl group elimination step. The hydroxylation step is found to be the rate-determining step with an energy barrier of 26.3 kcal/mol under the B3LYP/cc-pVTZ//CHARMM22 level. Two possible pathways for the second oxidation step are carefully investigated. Our simulations indicate that an oxygen atom from the coenzyme FADHOOH is inserted into the product. In addition, the electrostatic influence of 17 individual residues and five neighboring water molecules on the rate-determining step was estimated. The results indicate that groups Gly132/Ala133/Leu134, Met375/Gln376 and a water fence play a key role in facilitating the rate-determining step. On the other hand, residues Leu160, Val161 and Ser162 are found to be critical to suppress the rate-determining step. Our results lead to further understanding of the detailed catalytic pathways for nitro sugar biosynthesis.     

A water-assisted nucleophilic mechanism utilized by BphD,the meta-cleavage product hydrolase in biphenyl degradation

As members of the α/β-hydrolase superfamily, Meta-cleavage product (MCP) hydrolases generally utilize a Ser-His-Asp catalytic triad to hydrolyze the cleavage of CC bond during the aerobic catabolism of aromatic compounds by bacteria. BphD is one kind of MCP hydrolase that catalyzes the hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to 2-hydroxypenta-2,4-dienoic acid (HPD) and benzoate. In this article, a combined quantum mechanics and molecule mechanics (QM/MM) approach has been employed to explore the reaction mechanism of BphD from Burkholderia xenovorans LB400. On the basis of the recently resolved crystal structures, three computational models have been constructed. Our calculation results reveal that BphD utilizes a water-assisted nucleophilic mechanism, which contains acylation and deacylation stages. In acylation reaction, an active site water molecule assists the proton transfer from Ser112 to the carbanion intermediate (substrate) by forming hydrogen bonds with Ser112 and His265, and this proton transfer is in concert with the nucleophilic attack of deprotonated Ser112 on the C6-carbonyl of substrate to form the acylated intermediate. In deacylation, the Asp237-His265 dyad acts as a general base to activate the hydrolytic water, whose nucleophilic attack leads to the collapses of acyl-enzyme intermediate. The acylation and deacylation process correspond to the highest energy barriers of 21.0 and 23.9 kcal/mol, respectively. During the catalytic reaction, the active site water and Asp237-His265 dyad play an important role for each elementary steps.     

Effects of protein flexibility on the site of metabolism prediction for CYP2A6 substrates

Structure-based prediction for the site of metabolism (SOM) of a compound metabolized by human cytochrome P450s (CYPs) is highly beneficial in drug discovery and development. However, the flexibility of the CYPs’ active site remains a huge challenge for accurate SOM prediction. Compared with other CYPs, the active site of CYP2A6 is relatively small and rigid. To address the impact of the flexibility of CYP2A6 active site residues on the SOM prediction for substrates, in this work, molecular dynamics (MD) simulations and molecular docking were used to predict the SOM of 96 CYP2A6 substrates. Substrates with known SOM were docked into the snapshot structures from MD simulations and the crystal structures of CYP2A6. Compared to the crystal structures, the protein structures obtained from MD simulations showed more accurate prediction for SOM. Our results indicated that the flexibility of the active site of CYP2A6 significantly affects the SOM prediction results. Further analysis for the 40 substrates with definite K_m values showed that the prediction accuracy for the low K_m substrates is comparable to that by ligand-based methods.     

Understanding the molecular basis of stability in Kunitz (STI) family of inhibitors in terms of a conserved core tryptophan residue: A theoretical investigation

β-trefoil is one of the superfolds among proteins. Important classes of proteins like Interleukins (ILs), FibroblastGrowth Factors (FGFs), Kunitz (STI) family of inhibitors etc. belong to this fold. Kunitz (STI) family of inhibitors of proteins possess a highly conserved and structurally important Trytophan 91 (W91) residue, which stitches the top layer of the barrel with the lid. In this article we have investigated the molecular insights of the involvement of this W91 residue in the stability and folding pathway of Kunitz (STI) family. Winged bean Chymotrypsin inhibitor (WCI), a member of Kunitz (STI) family was chosen as a model system for carrying out the work. Molecular dynamics (MD) simulations were run with a set of total six proteins, including wild type WCI (WT) & five mutants namely W91F, W91M, W91A, W91H and W91I. Among all of them the coordinates of four proteins were taken from their crystal structures deposited in the Protein Data Bank (PDB), where as the coordinates for the rest two was generated using in-silico modelling. Our results suggest that truly this W91 residue plays a determining role in stability and folding pathway of Kunitz (STI) family. The mutants are less stable and more susceptible to quicker unfolding at higher temperatures compared to the wild type WCI. These effects are most pronounced for the smallest mutants namely W91H and W91A, indicating more is the cavity created by mutation at W91 position more the proteins becomes unstable.     

Investigation of the rescue mechanism catalyzed by a nucleophile mutant of rice BGlu1

In the present study, the quantum mechanical/molecular mechanical (QM/MM) method was used to investigate the rescue mechanism of an E386G mutant as well as the glycosylation mechanism of the wild rice β-d-glucosidase. E386G mutant experiences an asynchronous collaborative process to glycosylate the anionic formate with an energy barrier of 22.6 kcal/mol, while the energy barrier is 25.9 kcal/mol for the wild complex. The low energy barrier of the mutated complex suggests that anionic formate might be a good nucleophile to attack the anomeric carbon atom. Both energy barriers can be lowered when the leaving departure releases from the active site, suggesting that the product release, rather than chemistry, contributes to the rate limiting in BGlu1 mutants. Structure analyses also indicate that the external nucleophile has little steric hindrance with pocket residues and adjusts freely to proceed the rescue mechanism of the mutated complex. Our calculations provide a guide for the selectivity of exogenous nucleophiles in the future study of β-glucosidase.     

Prediction of the receptor conformation for iGluR2 agonist binding: QM/MM docking to an extensive conformational ensemble generated using normal mode analysis

Highly flexible proteins constitute a significant challenge in molecular docking within the field of drug design. Depending on the efficacy of the bound ligand, the ligand-binding domain (LBD) of the ionotropic glutamate receptor iGluR2 adopts markedly different degrees of domain closure due to large-scale domain movements. With the purpose of predicting the induced domain closure of five known iGluR2 partial to full agonists we performed a validation study in which normal mode analysis (NMA) was employed to generate a 25-membered ensemble of iGluR2 LBD structures with gradually changing domain closures, followed by accurate QM/MM docking to the ensemble. Based on the docking scores we were able to predict the correct optimal degree of closure for each ligand within 1–3° deviation from the experimental structures. We demonstrate that NMA is a useful tool for reliable ensemble generation and that we are able to predict the ligand induced conformational change of the receptor through docking to such an ensemble. The described protocol expands and improves the information that can be obtained from computational docking when dealing with a flexible receptor.     

The inhibitory effect of helenalin on telomerase activity is attributed to the alkylation of the CYS445 residue: Evidence from QM/MM simulations

Enhanced telomerase activity is a hallmark in the majority of cancer cells. Thus, understanding the interactions between telomerase and its inhibitors is fundamentally important for the development of novel anticancer drugs without severe side effects. In this study, the covalent binding of helenalin to CYS445 of telomerase (PDB ID: 3DU6) was simulated using combined quantum chemical and molecular mechanical (QM/MM) methods. The results showed that the reaction was a reversible Michael-type addition and a hydrogen bond was formed between helenalin and the side chain of LYS416 of telomerase during the reaction procedure. The LYS416 residue is vital to telomere DNA recognition by interacting with DNA base through hydrogen bonds. The alkylation of CYS445 of telomerase by helenalin may interfere with the telomere DNA recognition at the telomerase active site, thus resulting in inhibition of the enzyme activity.     

Molecular modeling study on the effect of residues distant from the nucleotide-binding portion on RNA binding in Staphylococcus aureus Hfq

Hfq is an abundant RNA-binding bacterial protein that was first identified in E. coli as a required host factor for phage Qβ RNA replication. The pleiotrophic phenotype resulting from the deletion of Hfq predicates the importance of this protein. Two RNA-binding sites have been characterized: the proximal site which binds sRNA and mRNA and the distal site which binds poly(A) tails. Previous studies mainly focused on the key residues in the proximal site of the protein. A recent mutation study in E. coli Hfq showed that a distal residue Val43 is important for the protein function. Interestingly, when we analyzed the sequence and structure of Staphylococcus aureus Hfq using the CONSEQ server, the results elicited that more functional residues were located far from the nucleotide-binding portion (NBP). From the analysis seven individual residues Asp9, Leu12, Glu13, Lys16, Gln31, Gly34 and Asp40 were selected to investigate the conformational changes in Hfq–RNA complex due to point mutation effect of those residues using molecular dynamics simulations. Results showed a significant effect on Asn28 which is an already known highly conserved functionally important residue. Mutants D9A, E13A and K16A depicted effects on base stacking along with increase in RNA pore diameter, which is required for the threading of RNA through the pore for the post-translational modification. Further, the result of protein stability analysis by the CUPSAT server showed destabilizing effect in the most mutants. From this study we characterized a series of important residues located far from the NBP and provide some clues that those residues may affect sRNA binding in Hfq.     

Binding mode analysis,dynamic simulation and binding free energy calculations of the MurF ligase from Acinetobacter baumannii

MurF ligase catalyzes the final cytoplasmic step of bacterial peptidoglycan biosynthesis and, as such, is a validated target for therapeutic intervention. Herein, we performed molecular docking to identify putative inhibitors of Acinetobacter baumannii MurF (AbMurF). Based on comparative docking analysis, compound 114 (ethyl pyridine substituted 3-cyanothiophene) was predicted to potentially be the most active ligand. Computational pharmacokinetic characterization of drug-likeness of the compound showed it to fulfil all the parameters of Muegge and the MDDR rule. A molecular dynamic simulation of 114 indicated the complex to be stable on the basis of an average root mean square deviation (RMSD) value of 2.09 Å for the ligand. The stability of the complex was further supported by root mean square fluctuation (RMSF), beta factor and radius of gyration values. Analyzing the complex using radial distribution function (RDF) and a novel analytical tool termed the axial frequency distribution (AFD) illustrated that after simulation the ligand is positioned in close vicinity of the protein active site where Thr42 and Asp43 participate in hydrogen bonding and stabilization of the complex. Binding free energy calculations based on the Poisson-Boltzmann or Generalized–Born Surface Area Continuum Solvation (MM(PB/GB)SA) method indicated the van der Waals contribution to the overall binding energy of the complex to be dominant along with electrostatic contributions involving the hot spot amino acids from the protein active site. The present results indicate that the screened compound 114 may act as a parent structure for designing potent derivatives against AbMurF in specific and MurF of other bacterial pathogens in general.     

The two-stage recombination operator and its application to the multiobjective 0/1 knapsack problem: A comparative study

In this paper, we first propose a new recombination operator called the two-stage recombination and then we test its performance in the context of the multiobjective 0/1 knapsack problem (MOKP). The proposed recombination operator generates only one offspring solution from a selected pair of parents according to the following two stages. In the first stage, called genetic shared-information stage or similarity-preserving stage, the generated offspring inherits all parent similar genes (i.e., genes or decision variables having the same positions and the same values in both parents). In the second stage, called problem fitness-information stage, the parent non-similar genes (i.e., genes or decision variables having the same positions but different values regarding the two parents) are selected from one of the two parents using some fitness information. Initially, we propose two different approaches for the second stage: the general version and the restricted version. However, the application of the restricted version to the MOKP leads to an improved version which is more specific to this problem. The general and the MOKP-specific versions of the two-stage recombination are compared against three traditional crossovers using two well-known multiobjective evolutionary algorithms. Promising results are obtained. We also provide a comparison between the general version and the MOKP-specific version.     

A C++ program for retrieving land surface temperature from the data of Landsat TM/ETM+ band6

A C++ language-based software tool for retrieving land surface temperature (LST) from the data of Landsat TM/ETM+ band6 is developed. It has two main functional modules: (1) Three methods to compute the ground emissivity based on land use/cover classification image, NDVI image and the ratio values of vegetation and bare ground and (2) Converting digital numbers (DNs) from TM/ETM+ band6 to LST. In the software tool, Qin et al.'s mono-window algorithm and Jiménez-Muňoz and Sobrino's single channel algorithm are programmed to retrieve LST. It will be a useful software tool to study the thermal environment of ground surface or the energy balance between the ground and the bottom atmosphere by using the thermal band of Landsat TM/ETM+.