Roles of K151 and D180 in L‐2‐haloacid dehalogenase from Pseudomonas sp. YL: Analysis by molecular dynamics and ab initio fragment molecular orbital calculations

L ‐2‐haloacid dehalogenase (L ‐DEX) catalyzes the hydrolytic dehalogenation of L ‐2‐haloalkanoic acids to produce the corresponding D ‐2‐hydroxyalkanoic acids. This enzyme is expected to be applicable to the bioremediation of environments contaminated with halogenated organic compounds. We analyzed the reaction mechanism of L ‐DEX from Pseudomonas sp. YL (L ‐DEX YL) by using molecular modeling. The complexes of wild‐type L ‐DEX YL and its K151A and D180A mutants with its typical substrate, L ‐2‐chloropropionate, were constructed by docking simulation. Subsequently, molecular dynamics (MD) and ab initio fragment molecular orbital (FMO) calculations of the complexes were performed. The ab initio FMO method was applied at the MP2/6‐31G level to estimate interfragment interaction energies. K151 and D180, which are experimentally shown to be important for enzyme activity, interact particularly strongly with L ‐2‐chloropropionate, catalytic water, nucleophile (D10), and with each other. Our calculations suggest that K151 stabilizes substrate orientation and balances the charge around the active site, while D180 stabilizes the rotation of the nucleophile D10, fixes catalytic water around D10, and prevents K151 from approaching D10. Further, D180 may activate catalytic water on its own or with K151, S175, and N177. These roles are consistent with the previous results. Thus, MD and ab initio FMO calculations are powerful tools for the elucidation of the mechanism of enzymatic reaction at the molecular level and can be applied to other catalytically important residues. The results obtained here will play an important role in elucidating the reaction mechanism and rational design of L ‐DEX YL with improved enzymatic activity or substrate specificity. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2009     

Microwave propagation and scattering in a dense distribution of non-tenuous spheres: experiment and theory

Controlled laboratory experimental results of coherent microwave propagation through a random medium are reported. The medium consisted of layers of styrofoam with spherical glass beads embedded at predetermined random positions generated by computer. The magnitude and phase of the transmitted field was measured over the frequency range 18-20.4 GHz for media with volume fractional densities ranging from 0.5% to 11%. The results are compared with independent scattering, Foldy's approximation, and the quasicrystalline approximation (QCA) using the solution of the Percus-Yevick (PY) equation for the pair distribution function. The effects of a size distribution are included. Experimental results indicate that at low densities, the measured extinction rate increases linearly with concentration in agreement with independent scattering. As concentration further increases, the extinction curve turns convex and is lower than independent scattering. However, it is higher than that predicted by QCA-PY. Using the known particle positions the authors have also computed the pair correlation function and good agreement is obtained with the Percus-Yevick approximation.     

Determination of cadmium,zinc, and lead by non-dispersive atomic fluorescence spectrometry with a new graphite furnace atomizer

A new graphite furnace atomizer has been developed and applied to the determination of cadmium, zinc, and lead by non-dispersive atomic fluorescence spectrometry. A solar-blind photomultiplier, a lock-in amplifier, and microwave-excited electrodeless discharge lamps are used. The detection limits for cadmium, zinc, and lead in the non-dispersive atomic fluorescence mode are 1·10^?13g, 2·10^?13g, and 2·10^?11g, respectively, which are 20-, 10-, and 2-fold better than those in the atomic absorption mode. The analytical working curves are linear over about three decades of concentration from the detection limits.     

Flow-injection chemiluminescent determination of vanadium(IV) and total vanadium by means of catalysis on the periodate oxidation of purpurogallin

A flow-injection chemiluminescent (CL) method is proposed for the successive determination of nanogram levels of vanadium(IV) and total vanadium. The method is based on the catalytic effect of vanadium(IV) on the oxidation of purpurogallin by periodate to produce light emission at 4 °C. The presence of hydrogen carbonate enhanced the light emission arising from the vanadium(IV)-catalyzed reaction. Since vanadium(V) did not catalyze the CL reaction of purpurogallin, vanadium(V) was determined after being reduced to vanadium(IV) by using an on-line silver-reducing column. Calibration curves for vanadium(IV) and (V) were linear in the range 0.1–10 ng ml⁻¹ with sampling rate of about 50 h⁻¹. The limit of detection for signal-to-noise ratio of 2 was 0.05 ng ml⁻¹ and the relative standard deviations were 1.4 and 1.6% for ten determinations of 2.0 ng ml⁻¹ vanadium(IV) and (V), respectively. Interferences from metal ions could be eliminated by the use of O,O′-bis(2-aminoethyl)ethyleneglycol- N,N,N′,N′-tetraacetic acid and diphosphate as masking agents. The proposed method was successfully applied to the determination of vanadium(IV) and total vanadium in fresh water samples.     

Flow-injection spectrophotometry of vanadium by catalysis of the bromate oxidation of N,N'-bis(2-hydroxyl-3-sulfopropyl)-tolidine

A highly sensitive flow-injection method is proposed for the catalytic determination of vanadium(V) at sub-nanogram per milliliter levels using a new indicator reaction. The method is based on the catalytic effect of vanadium(V) on the bromate oxidation of N,N′-bis(2-hydroxyl-3-sulfopropyl)-tolidine. 1,2-Dihydroxybenzene-3,5-disulfonate was used as an activator in the vanadium(V)-catalyzed reaction and significantly enhanced the sensitivity of the method. Vanadium(V) in the range 0.01-3.0 ng ml⁻¹ was easily determined with sampling rate of about 30 h⁻¹. Vanadium(IV) could be also determined. The limit of detection (S/N=3) was 0.008 ng ml⁻¹ and the relative standard deviations were 1.4 and 1.6% for ten determinations of 0.2 ng ml⁻¹ vanadium(IV) and vanadium(V), respectively. Interferences from metal ions could be suppressed by the addition of ethylenediamine-N,N,N′,N′-tetrakis(methylenephosphonic acid) as a masking agent. The proposed method was successfully applied to the determination of vanadium in water samples.     

Synthesis and biological activities of 4-phenyl-5-pyridyl-1,3-thiazole derivatives as p38 MAP kinase inhibitors

A novel series of 4-phenyl-5-pyridyl-1,3-thiazole analogues possessing potent in vitro inhibitory activity against p38 mitogen-activated protein kinase and the release of tumor necrosis factor-alpha (TNF-alpha) from human monocytic THP-1 cells stimulated by lipopolysaccharide has been identified. Subsequent structure-activity relationship (SAR) studies and optimization for absorption, distribution, metabolism, and elimination (ADME) profiles led to the identification of compounds 7 g and 10b as orally active lead candidates that block the in vivo production of proinflammatory cytokine (TNF-alpha). In pharmacokinetic studies, compound 10b showed good oral administration in mice and demonstrated significant in vivo anti-inflammatory activity in an anti-collagen monoclonal antibody-induced arthritis mouse model (minimum effective dose (MED)=30 mg/kg). Further elucidation of this class of compounds may provide novel anti-inflammatory agents, such as anti-rheumatoid arthritis drugs.     

Theoretical analysis of binding specificity of influenza viral hemagglutinin to avian and human receptors based on the fragment molecular orbital method

The hemagglutinin (HA) protein of the influenza virus binds to the host cell receptor in the early stage of viral infection. A change in binding specificity from avian 2-3 to human 2-6 receptor is essential for optimal human-to-human transmission and pandemics. Therefore, it is important to reveal the key factors governing the binding affinity of HA-receptor complex at the molecular level for the understanding and prediction of influenza pandemics. In this work, on the basis of ab initio fragment molecular orbital (FMO) method, we have carried out the interaction energy analysis of HA-receptor complexes to quantitatively elucidate the binding specificity of HAs to avian and human receptors. To discuss the binding property of influenza HA comprehensively, a number of HAs from human H1, swine H1, avian H3 and avian H5 viruses were analyzed. We performed detailed investigations about the interaction patterns of complexes of various HAs and receptor analogues, and revealed that intra-molecular interactions between conserved residues in HA play an important role for HA-receptor binding. These results may provide a hint to understand the role of conserved acidic residues at the receptor binding site which are destabilized by the electrostatic repulsion with sialic acid. The calculated binding energies and interaction patterns between receptor and HAs are consistent with the binding specificities of each HA and thus explain the receptor binding mechanism. The calculated results in the present analysis have provided a number of viewpoints regarding the models for the HA-receptor binding specificity associated with mutated residues. Examples include the role of Glu190 and Gln226 for the binding specificity of H5 HA. Since H5 HA has not yet been adapted to human receptor and the mechanism of the specificity change is unknown, this result is helpful for the prediction of the change in receptor specificity associated with forthcoming possible pandemics.     

Reversible O-O bond cleavage and formation of a peroxo moiety of a peroxocarbonate ligand mediated by an iron(III) complex

A mononuclear iron(III) complex containing a peroxocarbonate ligand, [Fe(qn)2(O2C(O)O)]- (qn = quinaldinate), underwent the reversible O-O bond cleavage and reformation of the peroxo group via the formation of FeIV=O or FeV=O species, which was confirmed by the resonance Raman and ESI-TOF/MS measurements.     

Growth enhancement of UF5 nano-particles assisted byα-ray ionization

It was demonstrated experimentally for the first time that particle growth of photoproduced UFS was enhanced by ionizing radiation from an -emitting²⁴¹Am source. A gas mixture of UF₆, CH₄, and Ar in a reaction cell was irradiated with a single shot of a KrF laser at 248 nm to form UF5 particles. Transmission electron microscope images of the samples indicated that the average size of UF₅ particles produced without the -ray source was independent of the laser energy. UFS particles produced with the -ray source were found to be much larger in size than those produced without the a-ray source, and the enhancement of particle growth was marked when the laser energy was low. An explanation was attempted for the anomalous behaviors of particle size distribution from the viewpoint of competition between the second-order coagulation rate and the rate of diffusion from the reaction zone.