In silico studies of the mechanism of methanol oxidation by quinoprotein methanol dehydrogenase

The mechanism of bacterial methanol dehydrogenase involves hydride equivalent transfer from substrate to the ortho-quinone PQQ to provide a C5-reduced intermediate that subsequently rearranges to the hydroquinone PQQH(2). We have studied the PQQ reduction by molecular dynamic (MD) simulations in aqueous solution. Among the five simulated structures, either Asp297 or Glu171 or both are ionized. Reasonable structures are obtained only when both carboxyl groups are ionized. This is not unexpected since the kinetic pH optimum is 9.0. In the structure of the enzyme.PQQ.HOCH(3) complex, the hydrogen bonded Glu171-CO(2)(-).H-OCH(3) is in a position to act as a general base catalyst for hydride equivalent transfer to C5 of PQQ. We thus suggest that Glu171 plays the role of general base catalyst in PQQ reduction rather than Asp297 as previously suggested. The reduction is assisted by Arg324, which hydrogen bonds to the ortho-quinone moiety of PQQ. The rearrangement of the C5-reduced intermediate to provide hydroquinone PQQH(2) is also assisted by proton abstraction by Glu171-CO(2)(-) and the continuous hydrogen bonding of Arg324 throughout the entire reaction. These features as well as the mapping of the channel for substrate and water into the active site entrance are the observations of major importance.     

Reaction mechanism of soluble epoxide hydrolase: insights from molecular dynamics simulations

Molecular dynamics simulations have been performed to gain insights into the catalytic mechanism of the hydrolysis of epoxides to vicinal diols by soluble epoxide hydrolase (sEH). The binding of a substrate, 1S,2S-trans-methylstyrene oxide, was studied in two conformations in the active site of the enzyme. It was found that only one is likely to be found in the active enzyme. In the preferred conformation the phenyl group of the substrate is pi-sandwiched between two aromatic residues, Tyr381 and His523, whereas the other conformation is pi-stacked with only one aromatic residue, Trp334. Two simulations were carried out to 1 ns for each conformation to evaluate the protonation state of active site residue His523. It was found that a protonated histidine is essential for keeping the active site from being disrupted. Long time scale, 4 ns, molecular dynamics simulation was done for the structure with the most likely combination of binding conformation and protonation state of His523. Near Attack Conformers (NACs) are present 5.3% of the time and nucleophilic attack on either epoxide carbon atom, approximately 75% on C(1) and approximately 25% on C(2), is found. A maximum of one hydrogen bond between the epoxide oxygen and either of the active site tyrosines, Tyr465 and Tyr381, is present, in agreement with experimental mutagenesis results that reveal a slight loss in activity if one tyrosine is mutated and essential loss of all activity upon double mutation of the two tyrosines in question. It was found that a hydrogen bond from Tyr465 to the substrate oxygen is essential for controlling the regioselectivity of the reaction. Furthermore, a relationship between the presence of this hydrogen bond and the separation of reactants was found. Two groups of amino acid segments were identified each as moving collectively. Furthermore, an overall anti-correlation was found between the movements of these two individually collectively moving groups, made up by parts of the cap-region, including the two tyrosines, and the site of the catalytic triad, respectively. This overall anti-correlated collective domain motion is, perhaps, involved in the conversion of E.NAC to E.TS.     

In silico model for P-glycoprotein substrate prediction: insights from molecular dynamics and in vitro studies

P-glycoprotein (P-gp) is a plasma membrane efflux transporter belonging to ATP-binding cassette superfamily, responsible for multidrug resistance in tumor cells. Over-expression of P-gp in cancer cells limits the efficacy of many anticancer drugs. A clear understanding of P-gp substrate binding will be advantageous in early drug discovery process. However, substrate poly-specificity of P-gp is a limiting factor in rational drug design. In this investigation, we report a dynamic trans-membrane model of P-gp that accurately identified the substrate binding residues of known anticancer agents. The study included homology modeling of human P-gp based on the crystal structure of C. elegans P-gp, molecular docking, molecular dynamics analyses and binding free energy calculations. The model was further utilized to speculate substrate propensity of in-house anticancer compounds. The model demonstrated promising results with one anticancer compound (NSC745689). As per our observations, the molecule could be a potential lead for anticancer agents devoid of P-gp mediated multiple drug resistance. The in silico results were further validated experimentally using Caco-2 cell lines studies, where NSC745689 exhibited poor permeability (P _app 1.03 ± 0.16 × 10^?6 cm/s) and low efflux ratio of 0.26.     

Polar solvation dynamics of lysozyme from molecular dynamics studies

The solvation dynamics of a protein are believed to be sensitive to its secondary structures. We have explored such sensitivity in this article by performing room temperature molecular dynamics simulation of an aqueous solution of lysozyme. Nonuniform long-time relaxation patterns of the solvation time correlation function for different segments of the protein have been observed. It is found that relatively slower long-time solvation components of the α-helices and β-sheets of the protein are correlated with lower exposure of their polar probe residues to bulk solvent and hence stronger interactions with the dynamically restricted surface water molecules. These findings can be verified by appropriate experimental studies.     

Biochemical studies of soluble and immobilized aldehyde dehydrogenase from yeast

Aldehyde dehydrogenase from baker’s yeast was purified to homogeneity. The soluble and immobilized forms of this enzyme were characterized and compared biochemically. These included steady state kinetics, stability study, fluorescence, and NMR spectroscopy. Evidence of the coenzyme binding to this enzyme in the absence of aldehyde substrates was obtained by fluorescence and NMR studies. A significant quenching of protein fluorescence was observed upon the addition of coenzymes to the aldehyde-free enzyme solution. Significant shifts and broadening of coenzyme proton resonances in the presence of enzyme also indicate the enzyme-coenzyme interactions in the aldehyde-free solution. the enzyme was immobilized on glass beads by three different methods. The immobilized enzyme was found to exhibit physical and biochemical properties similar to those of the soluble enzyme. A system in which the immobilized alcohol, aldehyde, and steriod dehydrogenases are included in an enzyme reactor is described.     

Enzymatic GTP hydrolysis: insights from an ab initio molecular dynamics study

Ab initio methods were used to shed light on fundamental aspects of the enzymatic mechanism of guanosine triphosphate hydrolysis in the Cdc42/Cdc42GAP complex. The calculations focused on the nucleophilic addition of the catalytic water molecule to the gamma-phosphate phosphorus atom. A large model system was required to correctly reproduce the electrostatic properties on the active site. The model turned out to reproduce most of the electrostatic field of the biological complex at the reactants. Our calculations established the H-bond pattern of the catalytic water (WAT), which turned out to interact with Q61 and T35, in the most stable conformation. This ruled out the possibility that the catalytic water transferred its proton directly to the gamma-phosphate. Furthermore, the calculations suggested that the electronic structure of WAT was very different from that in the bulk. Finally, this study showed that during the reaction, WAT transferred a proton to Gln61, consistent with the available X-ray data on a transition-state analogue/enzyme complex(19) and with the decrease of activity in the Q61E mutant.     

Aqua ions-graphene interfacial and confinement behavior: insights from isobaric-isothermal molecular dynamics

We carry out a systematic microstructural characterization of the solid-fluid interface (SFI) of water and simple metal chloride aqueous solutions in contact with a free-standing plate or with two such plates separated by an interplate distance 0 ≤ h (?) ≤ 30 at ambient conditions via isothermal-isobaric molecular dynamics. With this characterization, we target the interrogation of the system in search for answers to fundamental questions regarding the structure of the "external" and "internal" (confined) SFIs, the effect of the differential hydration behavior among species, and its link to species expulsion from confinement. For water at ambient conditions, we found that the structure of the "external" SFIs is independent of the interplate distance h in the range 0 ≤ h (?) ≤ 30, that is, the absence of wall-mediated correlation effects between "external" and "internal" SFIs, and that for h < 9 ? the slit-pores dewet. Moreover, we observed a selective expulsion of ions caused by the differential hydration between the anion and the cations with a consequent charging of the slit-pore. All these observations were interpreted in terms of the axial profiles for precisely defined order parameters, including tetrahedral configuration, hydrogen bonding, and species coordination numbers.     

Probing the neutral graphene-ionic liquid interface: insights from molecular dynamics simulations

We study basic mechanisms of the interfacial layer formation at the neutral graphite monolayer (graphene)-ionic liquid (1,3-dimethylimidazolium chloride, [dmim][Cl]) interface by fully atomistic molecular dynamics simulations. We probe the interface area by a spherical probe varying the charge (-1e, 0, +1e) as well as the size of the probe (diameter 0.50 nm and 0.38 nm). The molecular modelling results suggest that: there is a significant enrichment of ionic liquid cations at the surface. This cationic layer attracts Cl(-) anions that leads to the formation of several distinct ionic liquid layers at the surface. There is strong asymmetry in cationic/anionic probe interactions with the graphene wall due to the preferential adsorption of the ionic liquid cations at the graphene surface. The high density of ionic liquid cations at the interface adds an additional high energy barrier for the cationic probe to come to the wall compared to the anionic probe. Qualitatively the results from probes with diameter 0.50 nm and 0.38 nm are similar although the smaller probe can approach closer to the wall. We discuss the simulation results in light of available experimental data on the interfacial structure in ionic liquids.     

Unfolding the conformational behavior of peptide dendrimers: insights from molecular dynamics simulations

We present here the first comprehensive structural characterization of peptide dendrimers using molecular simulation methods. Multiple long molecular dynamics simulations are used to extensively sample the conformational preferences of five third-generation peptide dendrimers, including some known to bind aquacobalamine. We start by analyzing the compactness of the conformations thus sampled using their radius of gyration profiles. A more detailed analysis is then performed using dissimilarity measures, principal coordinate analysis, and free energy landscapes, with the aim of identifying groups of similar conformations. The results point to a high conformational flexibility of these molecules, with no clear "folded state", although two markedly distinct behaviors were found: one of the dendrimers displayed mostly compact conformations clustered into distinct basins (rough landscape), while the remaining dendrimers displayed mainly noncompact conformations with no significant clustering (downhill landscape). This study brings new insight into the conformational behavior of peptide dendrimers and may provide better routes for their functional design. In particular, we propose a yet unsynthesized peptide dendrimer that might exhibit enhanced ability to coordinate aquocobalamin.     

Nanoparticle dispersion and aggregation in polymer nanocomposites: insights from molecular dynamics simulation

It is a great challenge to fully understand the microscopic dispersion and aggregation of nanoparticles (NPs) in polymer nanocomposites (PNCs) through experimental techniques. Here, coarse-grained molecular dynamics is adopted to study the dispersion and aggregation mechanisms of spherical NPs in polymer melts. By tuning the polymer-filler interaction in a wide range at both low and high filler loadings, we qualitatively sketch the phase behavior of the PNCs and structural spatial organization of the fillers mediated by the polymers, which emphasize that a homogeneous filler dispersion exists just at the intermediate interfacial interaction, in contrast with traditional viewpoints. The conclusion is in good agreement with the theoretically predicted results from Schweizer et al. Besides, to mimick the experimental coarsening process of NPs in polymer matrixes (ACS Nano 2008, 2, 1305), by grafting polymer chains on the filler surface, we obtain a good filler dispersion with a large interparticle distance. Considering the PNC system without the presence of chemical bonding between the NPs and the grafted polymer chains, the resulting good dispersion state is further used to investigate the effects of the temperature, polymer-filler interaction, and filler size on the filler aggregation process. It is found that the coarsening or aggregation process of the NPs is sensitive to the temperature, and the aggregation extent reaches the minimum in the case of moderate polymer-filler interaction, because in this case a good dispersion is obtained. That is to say, once the filler achieves a good dispersion in a polymer matrix, the properties of the PNCs will be improved significantly, because the coarsening process of the NPs will be delayed and the aging of the PNCs will be slowed.     

How natural materials remove heavy metals from water: mechanistic insights from molecular dynamics simulations

Water pollution by heavy metals is of increasing concern due to its devastating effects on the environment and on human health. For the removal of heavy metals from water sources, natural materials, such as spent-coffee-grains or orange/banana/chestnut peels, appear to offer a potential cheap alternative to more sophisticated and costly technologies currently in use. However, in order to employ them effectively, it is necessary to gain a deeper understanding – at the molecular level – of the heavy metals-bioorganic-water system and exploit the power of computer simulations. As a step in this direction, we investigate via atomistic simulations the capture of lead ions from water by hemicellulose – the latter being representative of the polysaccharides that are common components of vegetables and fruit peels − as well as the reverse process. A series of independent molecular dynamics simulations, both classical and ab initio, reveals a coherent scenario which is consistent with what one would expect of an efficient capture, i.e. that it be fast and irreversible: (i) binding of the metal ions via adsorption is found to happen spontaneously on both carboxylate and hydroxide functional groups; (ii) in contrast, metal ion desorption, leading to solvation in water, involves sizable free-energy barriers.

We investigate via atomistic simulations the capture of lead ions from water by hemicellulose – as representative of the polysaccharides that are common components of vegetables and fruit peels – and the reverse process.     

Substrate-binding in quinoprotein ethanol dehydrogenase from pseudomonas aeruginosa studied by electron paramagnetic resonance at 94 GHz

Pyrroloquinoline quinone (2,7,9-tricarboxypyrroloquinoline quinone, PQQ) is one of several quinone cofactors that is utilized in a class of dehydrogenases known as quinoproteins. In this contribution, we have used continuous-wave high-field/high-frequency electron paramagnetic resonance (EPR) at 94 GHz (W-band) to study substrate binding in ethanol dehydrogenase (QEDH) from Pseudomonas aeruginosa, taking advantage of the fact that the enzyme is isolated with a substantial proportion of the PQQ cofactor in the paramagnetic semiquinone form. In the substrate-free enzyme, the principal values of the g-tensor, obtained by spectral simulation are: gx = 2.00585(2), gy = 2.00518(2), and gz = 2.00212(2), giving giso = 2.00438(2). All three principal values of the g-tensor decrease when ethanol is bound to the protein: gx = 2.00574(2), gy = 2.00511(2), and gz = 2.00207(2), giving giso = 2.00431(2). The results represent the first direct evidence for the tight binding of an alcohol to a PQQ-dependent alcohol dehydrogenase and show that ethanol also binds to the enzyme even when the PQQ cofactor is in the semiquinone form. The decrease in g is consistent with an increase in polarity in the immediate vicinity of the PQQ cofactor and probably reflects a changed geometry of the PQQ-Ca2+ complex when ethanol binds.     

Reactivation of a palladium catalyst during glucose oxidation by molecular oxygen

Kinetic data of glucose oxidation in aqueous solution catalyzed by heterogeneous palladium catalyst are presented under conditions of the catalyst reversible deactivation by oxygen. Measurements were run in a semi-continuous stirred tank reactor at 30°C and atmospheric pressure in kinetic regime. Effect of the reaction mixture composition on the reaction rate is presented. The catalyst activation/reactivation technique is discussed and optimized. Relation between optimal activation time and glucose concentration was defined.     

Separation of amino acids in glucose isomerase crystal: Insight from molecular dynamics simulations

The separation of amino acids (Arg, Phe and Trp) in a liquid chromatography is investigated using molecular dynamics simulations. A bioorganic nanoporous material – glucose isomerase crystal – is used as the stationary phase and water as the mobile phase. The transport velocities of amino acids decrease in the order Arg > Phe > Trp, consistent with experimental measurement. The elution order is not affected by the solute concentration or by the flowing rate of mobile phase. Arg is highly hydrophilic and charged, interacts with water the most strongly, and thus moves with flowing water the fastest. Trp has the largest van der Waals volume and encounters the largest steric hindrance, leading to the slowest velocity. From the number distributions of amino acids around protein surface, Trp and Phe are found to stay closer to protein than Arg. The solvent-accessible surface areas of amino acids and the numbers of hydrogen bonds between amino acids and water further elucidate the observed velocity difference. The simulation results provide useful microscopic insight into the retention mechanisms in chromatographic separation process and suggest that glucose isomerase crystal has the capability to separate amino acids.     

Charge-state dependent compaction and dissociation of protein complexes: insights from ion mobility and molecular dynamics

Collapse to compact states in the gas phase, with smaller collision cross sections than calculated for their native-like structure, has been reported previously for some protein complexes although not rationalized. Here we combine experimental and theoretical studies to investigate the gas-phase structures of four multimeric protein complexes during collisional activation. Importantly, using ion mobility-mass spectrometry (IM-MS), we find that all four macromolecular complexes retain their native-like topologies at low energy. Upon increasing the collision energy, two of the four complexes adopt a more compact state. This collapse was most noticeable for pentameric serum amyloid P (SAP) which contains a large central cavity. The extent of collapse was found to be highly correlated with charge state, with the surprising observation that the lowest charge states were those which experience the greatest degree of compaction. We compared these experimental results with in vacuo molecular dynamics (MD) simulations of SAP, during which the temperature was increased. Simulations showed that low charge states of SAP exhibited compact states, corresponding to collapse of the ring, while intermediate and high charge states unfolded to more extended structures, maintaining their ring-like topology, as observed experimentally. To simulate the collision-induced dissociation (CID) of different charge states of SAP, we used MS to measure the charge state of the ejected monomer and assigned this charge to one subunit, distributing the residual charges evenly among the remaining four subunits. Under these conditions, MD simulations captured the unfolding and ejection of a single subunit for intermediate charge states of SAP. The highest charge states recapitulated the ejection of compact monomers and dimers, which we observed in CID experiments of high charge states of SAP, accessed by supercharging. This strong correlation between theory and experiment has implications for further studies as well as for understanding the process of CID and for applications to gas-phase structural biology more generally.     

Mechanism of acetylcholinesterase inhibition by fasciculin: a 5-ns molecular dynamics simulation

Our previous molecular dynamics simulation (10 ns) of mouse acetylcholinesterase (EC 3.1.1.7) revealed complex fluctuation of the enzyme active site gorge. Now we report a 5-ns simulation of acetylcholinesterase complexed with fasciculin 2. Fasciculin 2 binds to the gorge entrance of acetylcholinesterase with excellent complementarity and many polar and hydrophobic interactions. In this simulation of the protein-protein complex, where fasciculin 2 appears to sterically block access of ligands to the gorge, again we observe a two-peaked probability distribution of the gorge width. When fasciculin is present, the gorge width distribution is altered such that the gorge is more likely to be narrow. Moreover, there are large increases in the opening of alternative passages, namely, the side door (near Thr 75) and the back door (near Tyr 449). Finally, the catalytic triad arrangement in the acetylcholinesterase active site is disrupted with fasciculin bound. These data support that, in addition to the steric obstruction seen in the crystal structure, fasciculin may inhibit acetylcholinesterase by combined allosteric and dynamical means. Additional data from these simulations can be found at http://mccammon.ucsd.edu/.     

Computational study of ketosteroid isomerase: insights from molecular dynamics simulation of enzyme bound substrate and intermediate

Delta(5)-3-Ketosteroid Isomerase (KSI) catalyzes the isomerization of 5,6-unsaturated ketosteroids to their 4,5-unsaturated isomers at a rate approaching the diffusion limit. The isomerization reaction follows a two-step general acid-base mechanism starting with Asp38-CO(2)(-) mediated proton abstraction from a sp(3)-hybridized carbon atom, alpha to carbonyl group, providing a dienolate intermediate. In the second step, Asp38-CO(2)H protonates the C6 of the intermediate providing a 4,5-unsaturated ketosteroid. The details of the mechanism have been highly controversial despite several experimental and computational studies of this enzyme. The general acid-base catalysis has been proposed to involve either a catalytic diad or a cooperative hydrogen bond mechanism. In this paper, we report our results from the 1.5 nanosecond molecular dynamics (MD) simulation of enzyme bound natural substrate (E.S) and enzyme bound intermediate (E.In) solvated in a TIP3P water box. The final coordinates from our MD simulation strongly support the cooperative hydrogen bond mechanism. The MD simulation of E.S and E.In shows that both Tyr14 and Asp99 are hydrogen bonded to the O3 of the substrate or intermediate. The average hydrogen bonding distance between Tyr14-OH and O3 becomes shorter and exhibits less fluctuation on E.S --> E.In. We also observe dynamic motions of water moving in and out of the active site in the E.S structures. This free movement of water disappears in the E.In structures. The active site is shielded by hydrophobic residues, which come together and squeeze out the waters from the active site in the E.In complex.     

Protein structure and dynamics in ionic liquids. Insights from molecular dynamics simulation studies

We present in this work the first molecular simulation study of an enzyme, the serine protease cutinase from Fusarium solani pisi, in two ionic liquids (ILs): 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) and 1-butyl-3-methylimidazolium nitrate ([BMIM][NO(3)]). We tested different water contents in these ILs at room temperature (298 K) and high temperature (343 K), and we observe that the enzyme structure is highly dependent on the amount of water present in the IL media. We show that the enzyme is preferentially stabilized in [BMIM][PF6] at 5-10% (w/w) (weight of water over protein) water content at room temperature. [BMIM][PF6] renders a more nativelike enzyme structure at the same water content of 5-10% (w/w) as previously found for hexane, and the system displays a similar bell-shape-like dependence with the water content in the IL media. [BMIM][PF6] is shown to increase significantly the protein thermostability at high temperatures, especially at low hydration. Our analysis indicates that the enzyme is less stabilized in [BMIM][NO(3)] relative to [BMIM][PF6] at both temperatures, most likely due to the strong affinity of the [NO(3)]- anion toward the protein main chain. These findings are in accordance with the experimental knowledge for these two ionic liquids. We also show that these ILs "strip off" most of the water from the enzyme surface in a degree similar to that found for polar organic solvents such as acetonitrile, and that the remaining waters at the enzyme surface are organized in many small clusters.     

Mechanism of the oxidation of aromatic sulfides catalysed by a water soluble iron porphyrin

The oxygen atom transfer-electron transfer (ET) mechanistic dichotomy has been investigated in the oxidation of a number of aryl sulfides by H2O2 in acidic (pH 3) aqueous medium catalysed by the water soluble iron(III) porphyrin 5,10,15,20-tetraphenyl-21H,23H-porphine-p,p',p",p"'-tetrasulfonic acid iron(III) chloride (FeTPPSCl). Under these reaction conditions, the iron-oxo complex porphyrin radical cation, P+. Fe(IV)=O, should be the active oxidant. When the oxidation of a series of para-X substituted phenyl alkyl sulfides (X = OCH3, CH3, H, Br, CN) was studied the corresponding sulfoxides were the only observed product and the reaction yields as well as the reactivity were little influenced by the nature of X as well as by the bulkiness of the alkyl group. Labelling experiments using H(2)18O or H(2)18O2 clearly indicated that the oxygen atom in the sulfoxides comes exclusively from the oxidant. Moreover, no fragmentation products were observed in the oxidation of a benzyl phenyl sulfide whose radical cation is expected to undergo cleavage of the beta C-H and C-S bonds. These results would seem to suggest a direct oxygen atom transfer from the iron-oxo complex to the sulfide. However, competitive experiments between thioanisole (E degree = 1.49 V vs. NHE in H2O) and N,N-dimethylaniline (E degree = 0.97 V vs. NHE in H2O) resulted in exclusive N-demethylation, whereas the oxidation of N-methylphenothiazine (10, E degree = 0.95 V vs. NHE in CH3CN) and N,N-dimethyl-4-methylthioaniline (11, E degree = 0.65 V vs. NHE in H2O) produced the corresponding sulfoxide with complete oxygen incorporation from the oxidant. Since an ET mechanism must certainly hold in the reactions of 10 and 11, the oxygen incorporation experiments indicate that the intermediate radical cation, once formed, has to react with PFe(IV)=O (the reduced form of the iron-oxo complex which is formed by the ET step) in a fast oxygen rebound. Thus, an ET step followed by a fast oxygen rebound is also suggested for the other sulfides investigated in this work.