A detailed molecular model for human aromatase

Using a variety of techniques, including sequence alignment, secondary struucture prediction, molecular mechanics and molecular dynamics, we have constructed a model for the three-dimensional structure of P-450_AROM (human aromatase) based on that of P-450_cam, the only cytochrome P-450 enzyme for which crystal structure is known. The predicted structure is found to be in good agreement with current experimental data; both direct, from site-directed mutagenesis studies, and indirect, from the consideration of the structures and activities of known substrates and inhibitors.     

Effect of mass transfer limitations on the enzymatic kinetic resolution of epoxides in a two-liquid-phase system

Optically active epoxides can be obtained by kinetic resolution of racemic mixtures using enantioselective epoxide hydrolases. To increase the productivity of the conversion of sparingly aqueous soluble epoxides, we investigated the use of a two-phase aqueous/organic system. A kinetic model which takes into account interphase mass transfer, enzymatic reaction, and enzyme inactivation was developed to describe epoxide conversion in the system by the epoxide hydrolase from Agrobacterium radiobacter. A Lewis cell was used to determine model parameters and results from resolutions carried out in the Lewis cell were compared to model predictions to validate the model. It was found that n-octane is a biocompatible immiscible solvent suitable for use as the organic phase. Good agreement between the model predictions and experimental data was found when the enzyme inactivation rate was fitted. Simulations showed that mass transfer limitations have to be avoided in order to maximize the yield of enantiomerically pure epoxide. Resolution of a 39 g/L solution of racemic styrene oxide in octane was successfully carried out in an emulsion batch reactor to obtain (S)-styrene oxide in high enantiomeric excess (>95% e.e.) with a yield of 30%.     

Identification of hotspot regions of MurB oxidoreductase enzyme using homology modeling,molecular dynamics and molecular docking techniques

Despite the availability of effective chemotherapy and a moderately protective vaccine, new anti-tuberculosis agents are urgently needed to decrease the global incidence of tuberculosis (TB) disease. The MurB gene belongs to the bacterial cell wall biosynthesis pathway and is an essential drug target in Mycobacterium tuberculosis (Mtb) that has no mammalian counterparts. Here, we present an integrated approach involving homology modeling, molecular dynamics and molecular docking studies on Mtb-MurB oxidoreductase enzyme. A homology model of Mtb-MurB enzyme was built for the first time in order to carry out structure-based inhibitor design. The accuracy of the model was validated using different techniques. The molecular docking study on this enzyme was undertaken using different classes of well known MurB inhibitors. Estimation of binding free energy by docking analysis indicated the importance of Tyr155, Arg156, Ser237, Asn241 and His304 residues within the Mtb-MurB binding pocket. Our computational analysis is in good agreement with experimental results of site-directed mutagenesis. The present study should therefore play a guiding role in the experimental design of Mtb-MurB inhibitors for in vitro/in vivo analysis.     

The energetics of embryonic growth and development. I. Oxygen consumption, biomass growth, and heat production.

A quantitative phenomenological model to describe the relationships between biomass growth rate, oxygen consumption, and heat production in developing embryos has been developed and tested using a wide range of experimental data. The model employs generalized material and energy balances, principles of enzyme kinetics, and an overall metabolic model scheme based on known biochemical principles. The phosphorylation concentration ratio of ATP and ADP occurs naturally and becomes a significant parameter in the analysis. The model is applied to the growth of Escherichia coli, Oryzias latipes, chick spinal cord, and whole chicken eggs. Excellent agreement between the model and the experimental data is obtained. In a succeeding paper (Part II) environmental effects and growth efficiency are discussed.     

A molecular model for vesicle formation

A molecular model is proposed to explain vesicle formation. The model is based on a balance between elastic and ‘hydrophobic’ forces for various micelle and bilayer geometries in dilute aqueous solutions. It predicts bilayered disc-like transition structures and is in agreement with experimental data.     

Mechanical equilibrium,a prerequisite to unveil auxetic properties in molecular compounds

The unique physical properties of auxetic compounds make them very attractive materials. Nevertheless, no synthesised materials known to exhibit negative Poisson’s ratio at the molecular level have been made. One way to explore such compounds is to predict potential candidates prior to their synthesis. To achieve it, multi-atom simulation is a powerful predicting tool. However, the lack of existing systems means that the crucial step of validation cannot be carried out. This paper aims to provide a procedure to predict the auxeticity of proposed molecular systems. The strategy is based on first revealing the determinant step in reaching the mechanical equilibrium of existing isotropic compounds such as polymers, or organic glasses, to compute efficiently mechanical properties. The Poisson’s ratio is found in good agreement with experimental data. The procedure is thus modified to be applied to anisotropic compounds, liquid crystals. The agreement with experimental behaviour allowed us to extrapolate the procedure to potentially auxetic compounds, thus offering great opportunities to reveal auxetic properties prior to the synthesis of the molecules.     

Toward a mathematical model of the assembly and disassembly of membrane microdomains: comparison with experimental models

We study a model system in which lipid bilayers are created using variable (precisely known) proportions of phosphatidylcholine and cholesterol. The model membranes exhibit cholesterol-enriched microdomains that are analogous to the so-called "lipid rafts" that form in living cells. After briefly presenting some experimental results, we formulate and solve a novel mathematical model based on the Smoluchowski equations for coagulation and fragmentation. We present a comparison between the distribution of lipid-raft areas observed in experimental lipid bilayers, and that distribution predicted by the theoretical model. Excellent agreement between the experiments and theory is obtained, with minimal parameter fitting.     

Using molecular dynamics simulations on crambin to evaluate the suitability of different continuum dielectric and hydrogen atom models for protein simulations

Molecular dynamics simulations of enzymes with enough explicit waters of solvation to realistically account for solute-solvent interactions can burden the computational resources required to perform the simulation by more than two orders of magnitude. Since enzyme simulations even with an implicit solvation model can be imposing for a supercomputer, it is important to assess the suitability of different continuum dielectric models for protein simulations. A series of 100-picosecond molecular dynamics simulations were performed on the X-ray crystal structure of the protein crambin to examine how well computed structures, obtained using seven continuum dielectric and two hydrogen atom models, agreed with the X-ray structure. The best level of agreement between computed and experimental structures was obtained using a constant dielectric of 2 and the all-hydrogen model. Continuum dielectric models of 1, 1r, and 2r also led to computed structures in reasonably good agreement with the X-ray structure. In all cases, the all-hydrogen model gave better agreement than the united atom model, although, in one case, the difference was not significant. Dielectric models of 4, 80, and 4r with either hydrogen model yielded significantly poorer fits. It is especially noteworthy that the observed trends did not semiquantitatively converge until about 50 picoseconds into the simulations, suggesting that validation studies for protein calculations based on energy minimizations or short simulations should be viewed with caution.     

Model for kinetics of myosin-V molecular motors

A hand-over-hand model is presented for the processive movement of myosin-V based on previous biochemical experimental results and structural observations of nucleotide-dependent conformational changes of single-headed myosins. The model shows that the ADP-release rate of the trailing head is much higher than that of the leading head, thus giving a 1 : 1 mechanochemical coupling for the processive movement of the motor. It explains well the previous finding that some 36-nm steps consist of two substeps, while other 36-nm steps consist of no substeps. Using the model, the calculated kinetic behaviors of myosin-V such as the main and intermediate dwell time distributions, the load dependence of the average main and intermediate dwell time and the load dependence of occurrence frequency of the intermediate state under various nucleotide conditions show good quantitative agreement with previous experimental results.     

Structural and functional homology between periplasmic bacterial molecular chaperones and small heat shock proteins

Abstract The periplasmic Yersinia pestis molecular chaperone Caf1M belongs to a superfamily of bacterial proteins for one of which (PapD protein of Escherichia coli ) the immunoglobulin-like fold was solved by X-ray analysis. The N-terminal domain of Caf1M was found to share a 20% amino acid sequence identity with an inclusion body-associated protein IbpB of Escherichia coli . One of the regions that was compared, was 32 amino acids long, and displayed more than 40% identity, probability of random coincidence was 1.2 × 10⁻⁴. IbpB is involved in a superfamily of small heat shock proteins which fulfil the function of molecular chaperone. On the basis of the revealed homology, an immunoglobulin-like one-domain model of IbpB three-dimensional structure was designed which could be a prototype conformation of sHsp's. The structure suggested is in good agreement with the known experimental data obtained for different members of sHsp's superfamily.     

Homology modelling and molecular dynamics study of human fatty acid amide hydrolase

The three-dimensional (3D) model of the human fatty acid amide hydrolase (hFAAH) was constructed based on the crystal structure of the rat FAAH (PDB code 1MT5) in complex with a substrate using Modeller9v2 program. With the aid of molecular mechanics and molecular dynamics method, the last model was obtained and further assessed by Profile-3D, Prosa2003 and Procheck, which confirms that the refined model is reliable. Furthermore, the docking results of propofol and its structural analogue into the active site of hFAAH indicate that 2,6-di-sec-butyl phenol is a more preferred ligand than others, which is in good agreement with the experimental results. From the docking studies, we also suggest that Phe192, Ile238, Thr377, Leu380, Phe381, Phe388 and Leu404 in the hFAAH are seven important determinant residues in binding as they have strong van der Waal interactions with the ligand.     

Catalytic oxidation of alkenes by manganese(III) porphyrin-encapsulated Al, V, Si-mesoporous molecular sieves

Cationic manganese-porphyrin, [meso-tetrakis(4-trimethylammoniophenyl)porphyrinato]manganese(III) pentachloride (MnTAPP), has been prepared and encapsulated into mesoporous molecular sieves Al-MCM-41 and V-MCM-41, containing different amounts of Al and V, respectively. The catalytic activities of these heterogeneous materials were tested in the liquid phase oxidation of cyclohexene and styrene in acetonitrile with iodosylbenzene (PhIO) as oxygen source. Both types of catalysts were active in the oxidation reaction. MnTAPP encapsulated in Al-MCM-41 produces allylic oxidation products alone and no epoxide with styrene was found. However, it produces both epoxide and allylic oxidation products with cyclohexene. At the same time, MnTAPP encapsulated in V-MCM-41 produces epoxide as major product and little allylic oxidation product with styrene, while both epoxide and allylic oxidation products were obtained with cyclohexene. It is suggested that the regioselective effect is due to relatively more acidic Al-MCM-41 than V-MCM-41 which could make the CC bond unreactive towards epoxidation and produces allylic oxidation product. With increasing Al or V content in the support, the porphyrin loading was found to increase, which in turn increases the catalytic activity of the heterogeneous systems. The heterogeneous catalysts were reused for three times. The selectivity of these heterogeneous catalysts does not change appreciably even after three times of reusing, but their catalytic activity decreases marginally. This may be attributed to catalyst leaching and/or decomposition of MnTAPP complex under the reaction conditions.     

Vitamin K, 2,3-Epoxide And Quinone Reduction: Mechanism And Inhibition

The chemical and enzymatic pathways of vitamin K₁ epoxide and quinone reduction have been investigated. The reduction of the epoxide by thiols is known to involve a thiol-adduct and a hydroxy vitamin K enolate intermediate which eliminates water to yield the quinone. Sodium borohydride treatment resulted in carbonyl reduction generating relatively stable compounds that did not proceed to quinone in the presence of base. NAD(P)H:quinone oxidoreductase (DT-diaphorase. E.C. I.6.99.2) reduction of vitamin K to the hydroquinone was a significant process in intact microsomes. but 1/5th the rate of the dithiothreitol (DTT)-dependent reduction. No evidence was found for DT-diaphorase catalyzed reduction of vitamin K₁ epoxide, nor was it capable of mediating transfer of electrons from NADH to the microsomal epoxide reducing enzyme. Purified diaphorase reduced detergent- solubilized vitamin K, 10^?5 as rapidly as it reduced dichlorophenylindophenol(DCPIP). Reduction of 10 μM vitamin K, by200 μM NADH was not inhibited by 10μM dicoumarol. whereas DCPIP reduction was fully inhibited. In contrast to vitamin K, (menadione). vitamin K₁ (phylloquinone) did not stimulate microsomal NADPH consumption in the presence or absence of dicoumarol. DTT-dependent vitamin K epoxide reduction and vitamin K reduction were shown to be mutually inhibitory reactions. suggesting that both occur at the same enzymatic site. On this basis, a mechanism for reduction of the quinone by thiols is proposed. Both the DTT-dependent reduction of vitamin K₁ epoxide and quinone. and the reduction of DCPIP by purified DT-diaphorase were inhibited by dicoumarol, warfarin. lapachol. and sulphaquinoxaline     

CHIH-DFT determination of the molecular structure infrared spectra,UV spectra and chemical reactivity of three antitubercular compounds: Rifampicin,Isoniazid and Pyrazinamide

Three of the most frequent antitubercular agents employed against Mycobacterium tuberculosis are: Rifampicin, Isoniazid and Pyrazinamide. It has been proven that the use of these antitubercular agents together, shortens the treatment period from 12–18 months to 6 months [1]. In this work we use a new Density Functional Theory chemistry model called CHIH-DFT (Chihuahua-Heterocycles-Density Functional Theory) that reflects the mixture of Hartree Fock exchange and DFT exchange, according to a mixing parameter based on empirical rules suited for heterocyclic systems. This new chemistry model was used to calculate the molecular structure of these antitubercular compounds, as well as their infrared, UV spectra, chemical reactivity and electronic properties. The UV and infrared spectra were obtained by experimental techniques. The calculated molecular structure, UV and IR spectra values from CHIH-DFT were compared with experimentally obtained values and theoretical studies. These results are in good agreement with experimental and theoretical studies. We also predicted using the relative electrophilicity and relative nucleophilicity concepts as defined by Roy et al. [2] the chemical active sites for the three antitubercular compounds as well as their electronegativity, ionization potential, electron affinity, hardness, dipole moment, E_HOMO-E_LUMO gap energy, etc.      

Reactivity, secondary structure, and molecular topology of the Escherichia coli sulfite reductase flavodoxin-like domain

The flavodoxin-like domain, missing in the three-dimensional structure of the monomeric, simplified model of the Escherichia coli sulfite reductase flavoprotein component (SiR-FP), has now been expressed independently. This 168 amino acid protein was named SiR-FP18 with respect to its native molecular weight and represents the FMN-binding domain of SiR-FP. This simplified biological object has kept the main characteristics of its counterpart in the native protein. It could incorporate FMN exclusively and stabilize a neutral air-stable semiquinone radical. Both the radical and the fully reduced forms of SiR-FP18 were able to transfer their electrons to DCPIP or cytochrome c quantitatively. SiR-FP18 was able to form a highly stable complex with SiR-HP, the hemoprotein component of the sulfite reductase containing an iron-sulfur cluster coupled to a siroheme. In agreement with the postulated catalytic cycle of SiR-FP, only the fully reduced form of SiR-FP18 could transfer one electron to SiR-HP, the transferred electron being localized exclusively on the heme. As isolated SiR-FP18 has kept the main characteristics of the FMN-binding domain of the native protein, a structural analysis by NMR was performed in order to complete the partial structure obtained previously. Structural modeling was performed using sequence homologues, cytochrome P450 reductase (CPR; 29% identity) and bacterial cytochrome P450 (P450-BM3; 26% identity), as conformational templates. These sequences were anchored using common secondary structural elements identified from heteronuclear NMR data measured on the protein backbone. The resulting structural model was validated, and subsequently refined using residual (C(alpha)-C', N-H(N), and C'-H(N)) dipolar couplings measured in an anisotropic medium. The overall fold of SiR-FP18 is very similar to that of bacterial flavodoxins and of the flavodoxin-like domain in CPR or P450-BM3.     

Crystal morphology study of N,N'-diacetylchitobiose by molecular dynamics simulation

Bisphenoidal shape of α-N,N′-diacetylchitobiose (α-(GlcNAc)₂) monohydrate crystals was obtained from aqueous solution. Crystal morphology was studied by computer simulation. Theoretical morphologies calculated by classic models (a BFDH model, a surface free-energy method, and an AE model) deviated significantly from that from the experimental crystal habit. Therefore, a solvent effect was considered by introducing an interface layer model based on molecular dynamics simulation in order to bridge this gap. The results of the simulation showed that the calculated habit is much closer to the experiment, meaning that the interface layer model describes the solvent effect very well. This model may also be applied for other oligosaccharide systems to study the relationship among crystal morphology, crystal structure, and solvation effects.     

Anchoring effects in a wide binding pocket: the molecular basis of regioselectivity in engineered cytochrome P450 monooxygenase from B. megaterium

The molecular basis of regioselectivity of cytochrome P450 monooxygenases from Bacillus megaterium (CYP102A1) with its flexible and widely opened active site is still not well understood. In the present work (-)-alpha-pinene bound complexes with two triple mutants were modeled to elucidate the contribution of the three major factors that mediate selectivity: active site shape, protein flexibility, and chemical reactivity of the substrate. For the triple mutant A74G F87V L188Q (GVQ), one stable, productive conformation of the substrate (conformation I) was identified by multiple molecular dynamics simulations. The model predicts pinene epoxide as a major product (42% pinene oxide, 23% verbenol) which is in agreement with the experimental product profile (70% pinene oxide, 20% verbenol). In contrast, for the triple mutant A74G F87G L188Q (GGQ) two stable productive substrate conformations were identified (conformations IIa and IIb), and verbenol was predicted as major product (81% verbenol, 16% myrtenol), which is in agreement with experimental results (77% verbenol, 10% myrtenol). The effect of chemical reactivity of the substrate was demonstrated by comparison of (-)-alpha-pinene to its regioisomer (-)-beta-pinene, where the product profile is shifted from 68% pinocarveol and 32% myrtanal in mutant GVQ, to 40% pinocarveol and 60% myrtanal in mutant GGQ. Our results strongly suggest a major role of residue 87 in anchoring (-)-alpha-pinene during substrate binding which provides a simple and elegant rationalization of the dynamic structure of this enzyme-substrate complex.     

Temperature and configurational effects on the Young’s modulus of poly (methyl methacrylate): a molecular dynamics study comparing the DREIDING,AMBER and OPLS force fields

The effects of the configuration and temperature on the Young’s modulus of poly (methyl methacrylate) (PMMA) have been studied using molecular dynamics simulations. For the DREIDING force field under ambient temperatures, increasing the number of monomers significantly increases the modulus of isotactic and syndiotactic PMMA while the isotactic form has a greater modulus. The effects of temperature on the modulus of isotactic PMMA have been simulated using the DREIDING, AMBER, and OPLS force fields. All these force fields predict the effects of temperature on the modulus from 200 to 350 K that are in close agreement with experimental values, while at higher temperatures the moduli are greater than those measured. The glass transition temperature determined by the force fields, based on the variation of the modulus with temperature, is greater than the experimental values, but when obtained from a plot of the volume as a function of the temperature, there is closer agreement. The Young’s moduli calculated in this study are in closer agreement to the experimental data than those reported by previous simulations.     

Kinetics, statistics, and energetics of lipid membrane electroporation studied by molecular dynamics simulations

Membrane electroporation is the method to directly transfer bioactive substances such as drugs and genes into living cells, as well as preceding electrofusion. Although much information on the microscopic mechanism has been obtained both from experiment and simulation, the existence and nature of possible intermediates is still unclear. To elucidate intermediates of electropore formation by direct comparison with measured prepore formation kinetics, we have carried out 49 atomistic electroporation simulations on a palmitoyl-oleoyl-phosphatidylcholine bilayer for electric field strengths between 0.04 and 0.7 V/nm. A statistical theory is developed to facilitate direct comparison of experimental (macroscopic) prepore formation kinetics with the (single event) preporation times derived from the simulations, which also allows us to extract an effective number of lipids involved in each pore formation event. A linear dependency of the activation energy for prepore formation on the applied field is seen, with quantitative agreement between experiment and simulation. The distribution of preporation times suggests a four-state pore formation model. The model involves a first intermediate characterized by a differential tilt of the polar lipid headgroups on both leaflets, and a second intermediate (prepore), where a polar chain across the bilayer is formed by 3-4 lipid headgroups and several water molecules, thereby providing a microscopic explanation for the polarizable volume derived previously from the measured kinetics. An average pore radius of 0.47 ± 0.15 nm is seen, in favorable agreement with conductance measurements and electrooptical experiments of lipid vesicles.