Density functional theory calculations on two glycosides, namely, n-octyl-β-D-glucopyranoside (C(8)O-β-Glc) and n-octyl-β-D-galactopyranoside (C(8)O-β-Gal) were performed for geometry optimization at the B3LYP/6-31G level. Both molecules are stereoisomers (epimers) differing only in the orientation of the hydroxyl group at the C4 position. Thus it is interesting to investigate electronically the effect of the direction (axial/equatorial) of the hydroxyl group at the C4 position. The structure parameters of X-H∙∙∙Y intramolecular hydrogen bonds were analyzed, while the nature of these bonds and the intramolecular interactions were considered using the atoms in molecules (AIM) approach. Natural bond orbital analysis (NBO) was used to determine bond orders, charge and lone pair electrons on each atom and effective non-bonding interactions. We have also reported electronic energy and dipole moment in gas and solution phases. Further, the electronic properties such as the highest occupied molecular orbital, lowest unoccupied molecular orbital, ionization energy, electron affinity, electronic chemical potential, chemical hardness, softness and electrophilicity index, are also presented here for both C(8)O-β-Glc and C(8)O-β-Gal. These results show that, while C(8)O-β-Glc possess- only one hydrogen bond, C(8)O-β-Gal has two intramolecular hydrogen bonds, which further confirms the anomalous stability of the latter in self-assembly phenomena.
Studies related to the engineering of calcium binding sites of CGTase are limited. The calcium binding regions that are known for thermostability function were subjected to site-directed mutagenesis in this study. The starting gene-protein is a variant of CGTase Bacillus sp. G1, reported earlier and denoted as "parent CGTase" herein. Four CGTase variants (S182G, S182E, N132R and N28R) were constructed. The two variants with a mutation at residue 182, located adjacent to the Ca-I site and the active site cleft, possessed an enhanced thermostability characteristic. The activity half-life of variant S182G at 60 °C was increased to 94 min, while the parent CGTase was only 22 min. This improvement may be attributed to the formation of a shorter α-helix and the alleviation of unfavorable steric strains by glycine at the corresponding region. For the variant S182E, an extra ionic interaction at the A/B domain interface increased the half-life to 31 min, yet it reduced CGTase activity. The introduction of an ionic interaction at the Ca-I site via the mutation N132R disrupted CGTase catalytic activity. Conversely, the variant N28R, which has an additional ionic interaction at the Ca-II site, displayed increased cyclization activity. However, thermostability was not affected.
PpCHS is a member of the type III polyketide synthase family and catalyses the synthesis of the flavonoid precursor naringenin chalcone from p-coumaroyl-CoA. Recent research reports the production of pyrone derivatives using either hexanoyl-CoA or butyryl-CoA as starter molecule. The Cys-His-Asn catalytic triad found in other plant chalcone synthase predicted polypeptides is conserved in PpCHS. Site directed mutagenesis involving these amino acids residing in the active-site cavity revealed that the cavity volume of the active-site plays a significant role in the selection of starter molecules as well as product formation. Substitutions of Cys 170 with Arg and Ser amino acids decreased the ability of the PpCHS to utilize hexanoyl-CoA as a starter molecule, which directly effected the production of pyrone derivatives (products). These substitutions are believed to have a restricted number of elongations of the growing polypeptide chain due to the smaller cavity volume of the mutant's active site.
Shigella flexneri serotype 2a is a major public health concern in the developing and under-developed countries which contributes to shigellosis endemic and mortality. Thus, there is an urgent need for a rapid diagnostic test for effective therapy and disease management. Previous study showed that a ∼35 kDa antigenic protein from S. flexneri is a potential biomarker. We therefore modelled the three-dimensional structure of the antigen to probe its functionality which could aid in the development of an antigen-based diagnostic. Results showed that the antigen is a transmembrane protein consists of OmpA and OmpA-like domains. The OmpA domain is a beta-barrel embedded in the outer membrane with four surface-exposed extracellular loops. The OmpA-like domain is linked to the OmpA domain with a 17 amino acids linker and located in the periplasmic. Docking of peptidoglycan into the groove of OmpA-like domain might help in catalyzing the bacterial cell wall formation. Both domains are expected to be involved in the virulence, structural stability, pathogenesis and survival of Shigella thus made the 35 kDa protein a suitable shigellosis diagnostic biomarker. This structural elucidation will also enable a better identification of the epitope regions for the development of specific binders to the 35 kDa antigen.
Here a mass spectrometry-based platform for the analysis of glycoproteins is presented. Glycopeptides and released glycans are analyzed, the former by quadrupole orthogonal time-of-flight liquid chromatography/mass spectrometry (QoTOF LC/MS) and the latter by permethylation analysis using matrix-assisted laser desorption/ionization (MALDI)-TOF MS. QoTOF LC/MS analysis reveals the stochastic distribution of glycoforms at occupied sequons, and the latter provides a semiquantitative assessment of overall protein glycosylation. Hydrophilic interaction chromatography (HILIC) was used for unbiased enrichment of glycopeptides and was validated using five model N-glycoproteins bearing a wide array of glycans, including high-mannose, complex, and hybrid subtypes such as sulfo and sialyl forms. Sialyl and especially sulfated glycans are difficult to analyze because these substitutions are labile. The conditions used here allow detection of these compounds quantitatively, intact, and in the context of overall glycosylation. As a test case, we analyzed influenza B/Malaysia/2506/2004 hemagglutinin, a component of the 2006-2007 influenza vaccine. It bears 11 glycosylation sites. Approximately 90% of its glycans are high mannose, and 10% are present as complex and hybrid types, including those with sulfate. The stochastic distribution of glycoforms at glycosylation sites is revealed. This platform should have wide applications to glycoproteins in basic sciences and industry because no apparent bias for any glycoforms is observed.
Peroxisome Proliferator-Activated Receptor γ (PPARγ) activators have drawn great recent attention in the clinical management of type 2 diabetes mellitus, prompting several attempts to discover and optimize new PPARγ activators. With this in mind, we explored the pharmacophoric space of PPARγ using seven diverse sets of activators. Subsequently, genetic algorithm and multiple linear regression analysis were employed to select an optimal combination of pharmacophoric models and 2D physicochemical descriptors capable of accessing self-consistent and predictive quantitative structure-activity relationship (QSAR) (r2(71)=0.80, F=270.3, r2LOO=0.73, r2PRESS against 17 external test inhibitors=0.67). Three orthogonal pharmacophores emerged in the QSAR equation and were validated by receiver operating characteristic (ROC) curves analysis. The models were then used to screen the national cancer institute (NCI) list of compounds. The highest-ranking hits were tested in vitro. The most potent hits illustrated EC50 values of 15 and 224 nM.
A thermophilic Bacillus stearothermophilus F1 produces an extremely thermostable serine protease. The F1 protease sequence was used to predict its three-dimensional (3D) structure to provide better insights into the relationship between the protein structure and biological function and to identify opportunities for protein engineering. The final model was evaluated to ensure its accuracy using three independent methods: Procheck, Verify3D, and Errat. The predicted 3D structure of F1 protease was compared with the crystal structure of serine proteases from mesophilic bacteria and archaea, and led to the identification of features that were related to protein stabilization. Higher thermostability correlated with an increased number of residues that were involved in ion pairs or networks of ion pairs. Therefore, the mutants W200R and D58S were designed using site-directed mutagenesis to investigate F1 protease stability. The effects of addition and disruption of ion pair networks on the activity and various stabilities of mutant F1 proteases were compared with those of the wild-type F1 protease.
A linear quantitative structure activity relationship (QSAR) model is presented for predicting human immunodeficiency virus-1 (HIV-1) reverse transcriptase enzyme inhibition. The 2D QSAR and 3D-QSAR models were developed by stepwise multiple linear regression, partial least square (PLS) regression and k-nearest neighbor-molecular field analysis, PLS regression, respectively using a database consisting of 33 recently discovered benzoxazinones. The primary findings of this study is that the number of hydrogen atoms, number of (-NH2) group connected with solitary single bond alters the inhibition of HIV-1 reverse transcriptase. Further, presence of electrostatic, hydrophobic and steric field descriptors significantly affects the ability of benzoxazinone derivatives to inhibit HIV-1 reverse transcriptase. The selected descriptors could serve as a primer for the design of novel and potent antagonists of HIV-1 reverse transcriptase.
A capillary electrophoretic method for the separation of the enantiomers of both ofloxacin and ornidazole is described. Several parameters affecting the separation were studied, including the type and concentration of chiral selector, buffer pH, voltage and temperature. Good chiral separation of the racemic mixtures was achieved in less than 16 min with resolution factors Rs=5.45 and 6.28 for ofloxacin and ornidazole enantiomers, respectively. Separation was conducted using a bare fused-silica capillary and a background electrolyte (BGE) of 50 mM H(3)PO(4)-1 M tris solution; pH 1.85; containing 30 mg mL(-1) of sulfated-beta-cyclodextrin (S-beta-CD). The separation was carried out in reversed polarity mode at 25 degrees C, 18 kV, detection wavelength at 230 nm and using hydrodynamic injection for 15 s. Acceptable validation criteria for selectivity, linearity, precision, and accuracy were studied. The limits of detection (LOD) and limits of quantitation (LOQ) of the enantiomers (ofloxacin enantiomer 1 (OF-E1), ofloxacin enantiomer 2 (OF-E2), ornidazole enantiomer 1 (OR-E1) and ornidazole enantiomer 2 (OR-E2)) were (0.52, 0.46, 0.54, 0.89) and (1.59, 1.40, 3.07, 2.70) microg mL(-1), respectively. The proposed method was successfully applied to the assay of enantiomers of both ofloxacin and ornidazole in pharmaceutical formulations. The computational calculations for the enantiomeric inclusion complexes rationalized the reasons for the different migration times between the ofloxacin and ornidazole enantiomers.
One of the most important goals in bioinformatics is the ability to predict tertiary structure of a protein from its amino acid sequence. In this paper, new feature groups based on the physical and physicochemical properties of amino acids (size of the amino acids' side chains, predicted secondary structure based on normalized frequency of β-Strands, Turns, and Reverse Turns) are proposed to tackle this task. The proposed features are extracted using a modified feature extraction method adapted from Dubchak et al. To study the effectiveness of the proposed features and the modified feature extraction method, AdaBoost.M1, Multi Layer Perceptron (MLP), and Support Vector Machine (SVM) that have been commonly and successfully applied to the protein folding problem are employed. Our experimental results show that the new feature groups altogether with the modified feature extraction method are capable of enhancing the protein fold prediction accuracy better than the previous works found in the literature.
Japanese encephalitis virus (JEV) is the most important cause of epidemic encephalitis worldwide but its origin is unknown. Epidemics of encephalitis suggestive of Japanese encephalitis (JE) were described in Japan from the 1870s onwards. Four genotypes of JEV have been characterised and representatives of each genotype have been fully sequenced. Based on limited information, a single isolate from Malaysia is thought to represent a putative fifth genotype. We have determined the complete nucleotide and amino acid sequence of Muar strain and compared it with other fully sequenced JEV genomes. Muar was the least similar, with nucleotide divergence ranging from 20.2 to 21.2% and amino acid divergence ranging from 8.5 to 9.9%. Phylogenetic analysis of Muar strain revealed that it does represent a distinct fifth genotype of JEV. We elucidated Muar signature amino acids in the envelope (E) protein, including E327 Glu on the exposed lateral surface of the putative receptor binding domain which distinguishes Muar strain from the other four genotypes. Evolutionary analysis of full-length JEV genomes revealed that the mean evolutionary rate is 4.35 × 10(-4) (3.4906 × 10(-4) to 5.303 × 10(-4)) nucleotides substitutions per site per year and suggests JEV originated from its ancestral virus in the mid 1500s in the Indonesia-Malaysia region and evolved there into different genotypes, which then spread across Asia. No strong evidence for positive selection was found between JEV strains of the five genotypes and the E gene has generally been subjected to strong purifying selection.
A series of Zn(II)-Schiff bases I, II and III complexes were synthesized by reaction of o-phenylenediamine with 3-methylsalicylaldehyde, 4-methylsalicylaldehyde and 5-methylsalicylaldehyde. These complexes were characterized using FT-IR, UV-Vis, Diffuse reflectance UV-Vis, elemental analysis and conductivity. Complex III was characterized by XRD single crystal, which crystallizes in the triclinic system, space group P-1, with lattice parameters a=9.5444(2) Å, b=11.9407(2) Å, c=21.1732(3) Å, V=2390.24(7) Å(3), D ( c )=1.408 Mg m(-3), Z=4, F(000)=1050, GOF=0.981, R1=0.0502, wR2=0.1205. Luminescence property of these complexes was investigated in DMF solution and in the solid state. Computational study of the electronic properties of complex III showed good agreement with the experimental data.
A linear quantitative structure activity relationship (QSAR) model is presented for modeling and predicting the inhibition of HIV-1 integrase. The model was produced by using the stepwise multiple linear regression technique on a database that consists of 67 recently discovered 1,3,4-oxadiazole substituted naphthyridine derivatives. The developed QSAR model was evaluated for statistical significance and predictive power. The key conclusion of this study is that valence connectivity index order 1, lowest unoccupied molecular orbital and dielectric energy significantly affect the inhibition of HIV-1 integrase activity by 1,3,4-oxadiazole substituted naphthyridine derivatives. The selected physicochemical descriptors serve as a first guideline for the design of novel and potent antagonists of HIV-1 integrase.
The stability of biocatalysts is an important criterion for a sustainable industrial operation economically. T1 lipase is a thermoalkalophilic enzyme derived from Geobacillus zalihae strain T1 (T1 lipase) that was isolated from palm oil mill effluent (POME) in Malaysia. We report here the results of high temperatures molecular dynamics (MD) simulations of T1 lipase in explicit solvent. We found that the N-terminal moiety of this enzyme was accompanied by a large flexibility and dynamics during temperature-induced unfolding simulations which preceded and followed by clear structural changes in two specific regions; the small domain (consisting of helices alpha3 and alpha5, strands beta1 and beta2, and connecting loops) and the main catalytic domain or core domain (consisting of helices alpha6- alpha9 and connecting loops which located above the active site) of the enzyme. The results suggest that the small domain of model enzyme is a critical region to the thermostability of this organism.
A new Mn(III) complex, [MnCl(H(2)O)(L)].H(2)O.C(2)H(5)OH, where L=2,2'-[1,2-phenylenebis[nitrilomethylylidene]]bis(6-methoxyphenolate), has been synthesized and characterized by single-crystal X-ray diffraction. There is a good agreement between calculated and experimental structural data. The complex is crystallized in orthorhombic with space group Pbca. The Mn1 atom is coordinated with one Schiff base ligand, one water molecule and one chloride anion, forming a six-coordination number. The electronic and fluorescence spectra of the complex were also studied.
An all-atom level MD simulation in explicit solvent at high temperature is a powerful technique to increase our knowledge about the structurally important regions modulating thermal stability in thermenzymes. In this respect, two large-sized thermoalkalophilic enzymes from Bacillus stearothermophilus L1 (L1 lipase) and Geobacillus zalihae strain T1 (T1 lipase) are well-established representatives. In this paper, comparative results from temperature-induced MD simulations of both model systems at 300 K, 400 K and 500 K are presented and discussed with respect to identification of highly flexible regions critical to thermostability. From our MD simulation results, specific regions along the L1 lipase and T1 lipase polypeptide chain including the small domain and the main catalytic domain or core domain of both enzymes show a marked increase in fluctuations and dynamics followed by clear structural changes. Overall, the N-terminal moiety of both enzymes and their small domains exhibit hyper-sensitivity to thermal stress. The results appear to propose that these regions are critical in determining of the overall thermal stability of both organisms.
(1)H NMR evidence for direct coordination between the Ln(III) ion and the oxygen atoms of the pentaethylene glycol (EO5) ligand and the picrate anion (Pic) in [Ln(Pic)(2)(EO5)][Pic] {Ln=Ce and Nd} complexes are confirmed by single X-ray diffraction. No dissociation of Ln-O bonds in dimethyl sulfoxide-d solution was observed in NMR studies conducted at different temperatures ranging 25-100 degrees C. The Ln(III) ion was chelated to nine oxygen atoms from the EO5 ligand in a hexadentate manner and the two Pic anions in each bidentate and monodentate modes. Both compounds are isostructural and crystallized in monoclinic with space group P2(1)/c. Coordination environment around the Ce1 and Nd1 atoms can be described as tricapped trigonal prismatic and monocapped square antiprismatic geometries, respectively. The crystal packing of the complexes have stabilized by one dimensional (1D) chains along the [001] direction to form intermolecular O-Hcdots, three dots, centeredO and C-Hcdots, three dots, centeredO hydrogen bonding. The molar conductance of the complexes in DMSO solution indicated that both compounds are ionic. The complexes had a good thermal stability. Under the UV-excitation, these complexes exhibited the red-shift emission.
A cytotoxic bisindole alkaloid possessing an unprecedented structure in which two indole moieties are bridged by an aromatic spacer unit has been isolated from Alstonia angustifolia. The structure was established by analysis of the spectroscopic data and confirmed by X-ray diffraction analysis. A possible biogenetic pathway from pyrocatechuic acid and pleiocarpamine is presented.
A group of flavanones and their chalcones, isolated from Boesenbergia rotunda L., were previously reported to show varying degrees of noncompetitive inhibitory activities toward Dengue virus type 2 (Den2) protease. Results obtained from automated docking studies are in agreement with experimental data in which the ligands were shown to bind to sites other than the active site of the protease. The calculated K(i) values are very small, indicating that the ligands bind quite well to the allosteric binding site. Greater inhibition by pinostrobin, compared to the other compounds, can be explained by H-bonding interaction with the backbone carbonyl of Lys74, which is bonded to Asp75 (one of the catalytic triad residues). In addition, structure-activity relationship analysis yields structural information that may be useful for designing more effective therapeutic drugs against dengue virus infections.
Five new cytotoxic limonoids, erythrocarpines A-E (1-5), were isolated from the bark of Chisocheton erythrocarpus Hiern. Chemical structures, stereochemistry, and conformation were fully elucidated and characterized by 2D NMR, MS, and computational methods.