Identity of the quinone in Bacillus alcalophilus.

Every Bacillus species so far examined contains menaquinone as the sole quinone. In contrast, the alkalophilic Bacillus alcalophilus has been reported to be unusual in containing ubiquinone rather than menaquinone. In this communication, we demonstrate that B. alcalophilus, like all the other bacilli, contains menaquinone as the only quinone.     

The fate of the fusarium mycotoxin zearalenone in maize cell suspension cultures

The fusarium mycotoxin zearalenone was transformed in cell suspension cultures of Zea mays giving α- and β-zearalenol and the β-D-glu cos ides of zearalenone and α- and β-zearalenol. The structure of zearalenone-4-β-D-glucopyranoside was determined by liquid — chromatography-mass spectrometry and specific hydrolysis with β-glucosidase. α- and β-zearalenol and their glucosides were identified by co chromatography using tic and HPLC and glucosidase — treatment Up to 50% of the mycotoxin added was bound to a non extractable or “bound” residue fraction. After treating this residue by a sequential cell wall fractionation procedure, zearalenone was found to be bound mainly to starch, hemicellulose, and lignin fractions.     

Gene transfer is a major factor in bacterial evolution

Lateral gene transfer in four strains of Salmonella enterica has been assessed using genomic subtraction. Strain LT2 (subspecies I serovar Typhimurium) chromosomal DNA was used as target and subtracted by three subspecies I strains of serovars Typhimurium (S21), Muenchen (S71), Typhi (M229), and a subspecies V strain (M321). Data from probing random cosmids of LT2 DNA with preparations of the residual LT2 DNA after subtraction were used to estimate the amounts of LT2 DNA not able to hybridize to strains S21, S71, M229, and M321 to be in the range of 84-106, 191-355, 305-629, and 778-1,286 kb, respectively. Several lines of evidence indicate that most of this DNA is from genes not present in strain M321 and not from genes that have diverged in sequence. The amounts correlate with the divergence of the four strains as revealed by multilocus enzyme electrophoresis and sequence variation of housekeeping genes. Sequence of 39 of the fragments from the M321 subtracted residual LT2 DNA revealed only six inserts of known gene function with evidence of both gain and loss of genes during the development of S. enterica clones. Sixteen of the 39 segments have 45% or lower G+C content, below the species average, but over half are within the normal range for the species. We conclude that even within a species, clones may differ by up to 20% of chromosomal DNA, indicating a major role for lateral transfer, and that on the basis of G+C content, a significant proportion of the DNA is from distantly related species.      

Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and characterization of a new isochorismate synthase from Escherichia coli.

The first committed step in the biosynthesis of menaquinone (vitamin K2) is the conversion of chorismate to isochorismate, which is mediated by an isochorismate synthase encoded by the menF gene. This isochorismate synthase (MenF) is distinct from the entC-encoded isochorismate synthase (EntC) involved in enterobactin biosynthesis. MenF has been overexpressed under the influence of the T7 promoter and purified to homogeneity. The purified protein was found to have a molecular mass of 98 kDa as determined by gel filtration column chromatography on Sephacryl S-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a molecular mass of 48 kDa. Thus, the enzyme is a homodimer. The purified enzyme showed a pH optimum of 7.5 to 8.0 and a temperature optimum of 37 degrees C. The enzyme carries out the irreversible conversion of chorismate to isochorismate in the presence of Mg2+. The enzyme was found to have a Km of 195 +/- 23 microM and a k(cat) of 80 min(-1). In the presence of 30 mM beta-mercaptoethanol (BME), the k(cat) increased to 176 min(-1). The reducing agents BME and dithiothreitol stimulated the enzymatic activity more than twofold. Treatment of the enzyme with the cysteine-specific modifying reagent N-ethylmaleimide (NEM) resulted in the complete loss of activity. Preincubation of the enzyme with the substrate, chorismate, before NEM treatment resulted in complete protection of the enzyme from inactivation.     

Stability of enzymes in starving Arthrobacter crystallopoietes.

Cell-free extracts prepared from spherical and rod-shaped cells of Arthrobacter crystallopoietes were assayed for enzymes during various periods of starvation. The level of NADH oxidase dropped to 20 and 30%, respectively, in spherical and rod-shaped cells during the first 1 to 2 days of starvation and then remained constant for 9 days. Catalase activity decreased continuously and reached a low level in 9 days. Enzymes involved in glucose metabolism and the tricarboxylic acid cycle were stable for the duration of the experiment (about 1 week). Succinic dehydrogenase, fumarase and aconitase were stable during 21 days of starvation, which is the longest time enzymes have been shown to be stable in any bacterium under conditions of total starvation.     

Discovery of potent inhibitors for interleukin-2-inducible T-cell kinase: structure-based virtual screening and molecular dynamics simulation approaches

In our study, a structure-based virtual screening study was conducted to identify potent ITK inhibitors, as ITK is considered to play an important role in the treatment of inflammatory diseases. We developed a structure-based pharmacophore model using the crystal structure (PDB ID: 3MJ2) of ITK complexed with BMS-50944. The most predictive model, SB-Hypo1, consisted of six features: three hydrogen-bond acceptors (HBA), one hydrogen-bond donor (HBD), one ring aromatic (RA), and one hydrophobic (HY). The statistical significance of SB-Hypo1 was validated using wide range of test set molecules and a decoy set. The resulting well-validated model could then be confidently used as a 3D query to screen for drug-like molecules in a database, in order to retrieve new chemical scaffolds that may be potent ITK inhibitors. The hits retrieved from this search were filtered based on the maximum fit value, drug-likeness, and ADMET properties, and the hits that were retained were used in a molecular docking study to find the binding mode and molecular interactions with crucial residues at the active site of the protein. These hits were then fed into a molecular dynamics simulation to study the flexibility of the activation loop of ITK upon ligand binding. This combination of methodologies is a valuable tool for identifying structurally diverse molecules with desired biological activities, and for designing new classes of selective ITK inhibitors.

Molecular structure,vibrational spectra and HOMO,LUMO analysis of 4-piperidone by density functional theory and ab initio Hartree–Fock calculations

Vibrational frequencies and geometrical parameters of 4-piperidone (4-PID) in the ground state have been calculated by using the Hartree–Fock (HF) and density functional methods (B3LYP) with 6-311++G(d,p) and 6-311+G(3df,2p) basis sets. These methods are proposed as a tool to be applied in the structural characterisation of 4-PID (C₅H₉NO). The title molecule has C _s point group symmetry, thus providing useful support in the interpretation of experimental IR and Raman data. The DFT-B3LYP/6-311+G(3df,2p) calculations have been found more reliable than the ab initio HF/6-311++G(d,p) calculations for the vibrational study of 4-PID. The calculated highest occupied molecular orbital and lowest unoccupied molecular orbital energies show that charge transfer occurs within the molecule. The theoretical spectrograms for FT-IR and FT-Raman spectra of the title molecule have been constructed.