Hydrophobic aggregation and collective absorption of dioxin into lipid membranes: insights from atomistic simulations.

Dioxins are a highly toxic class of chlorinated aromatic chemicals. They have been extensively studied, but several molecular-level details of their action are still missing. Here we present molecular dynamics simulations of their absorption and diffusion through cell membranes. We show that, due to their hydrophobic character, dioxins can quickly penetrate into a lipid membrane, both as single molecules and as aggregates. We find clear evidence for their ability to accumulate in cell membranes. Our free energy calculations indicate that subsequent transport into the cell is unlikely to be a simple diffusive process.

Aminophosphonic acids and derivatives. Synthesis and biological applications.

In recent years, phosphonic acids and their derivatives have received increasing attention as analogues of a series of naturally occurring phosphates and as "bio-isosteric phosphorus analogues" of amino acids. Unlike a phosphate group, the phosphonate moiety is not readily hydrolyzed, in a biological environment, by the enzymes involved in the phosphate cleavage. This feature makes these compounds extremely useful in several applications, in metabolic regulation, in enhancement or inhibition studies, in the development of potential drugs against several metabolic disorders. The great potential of these compounds in biological applications resulted in an intense effort directed to the development of efficient synthetic methods for their preparation, with particular attention to stereoselective synthesis. The purpose of this review is to give an up-to-date account of the chemistry, the synthesis and the biological activity of aminophosphonic acids and their derivatives.

Dioxygenation of naphthalene by Pseudomonas fluorescens N3 dioxygenase: optimization of the process parameters.

The bioconversion of naphthalene to the 1,2-dihydro-1,2-dihydroxy derivative was performed in good yield using an Escherichia coli recombinant strain carrying Pseudomonas fluorescens N3 dioxygenase. However, the efficiency of such transformation is affected by many process parameters, and their optimization is essential to the scaling up of the process. The following process parameters were considered for optimization: cell concentration together with the corresponding glucose concentration (DCW/L); pH of medium; temperature; stirring speed; air flow; substrate concentration; Fe(2+) concentration; microelements concentration; reaction volume. We used a two-step multivariate experimental design to select important variables and assign them optimal values. The most significant parameters were selected by adopting a Plackett-Burman design, and were then correlated, using a full factorial design, with the experimental results. The experimental results illustrate that the optimized process of recombinant whole cell biotransformation in two-liquid phase systems enhances the naphthalene dihydrodiol yield threefold. This biotransformation opens the way to future experiments involving different substrates.

Characterization of Rhodococcus opacus R7, a strain able to degrade naphthalene and o-xylene isolated from a polycyclic aromatic hydrocarbon-contaminated soil.

Rhodococcus opacus R7 was isolated from a soil contaminated with polycyclic aromatic hydrocarbons for its ability to grow on naphthalene. The strain was also able to degrade o-xylene, the isomer of xylenes most recalcitrant to microbial degradation. The catabolic pathways for naphthalene and o-xylene were investigated by identification of metabolites in R. opacus R7 cultures performed with the two hydrocarbons and by evaluation of some enzymes involved in the metabolism of these compounds. 1,2-Dihydro-1,2-dihydroxynaphthalene, salicylic and gentisic acids were identified as metabolites in cultures exposed to naphthalene. This suggests that the degradation occurs through the dioxygenation of the aromatic ring with the formation of 1,2-dihydro-1,2-dihydroxynaphthalene, dehydrogenated to the corresponding 1,2-dihydroxy derivative which is further oxidized to salicylic acid, a key intermediate of naphthalene metabolism; this compound is converted to gentisic acid cleaved by a gentisate 1,2-dioxygenase. From R. opacus R7 cultures supplied with o-xylene, 2,3-dimethylphenol and 3,4-dimethylcatechol were observed. The pathway of o-xylene involves the monooxygenation of the benzene nucleus leading to dimethylphenol which is further metabolised to 3,4-dimethylcatechol, followed by a meta cleavage reaction, catalyzed by the catechol 2,3-dioxygenase. R. opacus R7 is the first strain thus far described both in Gram-negative and Gram-positive bacteria which has the ability to degrade both a polycyclic aromatic hydrocarbon such as naphthalene and a monocyclic aromatic hydrocarbon such as o-xylene.

Predicting toxicity: a mechanism of action model of chemical mutagenicity.

The increasing importance of theoretical studies for predicting toxicology has aroused the interest of many computational chemists. A new approach has been developed, based on studying at the molecular level two potential mechanisms of action that are related to compound mutagenicity. This approach is the first example that considers both the toxicant and the biological target molecules involved in the interaction. Using some calculated descriptors and a simulation of the interaction chemical, compounds can be classified. More important, the approach helps in understanding and explaining both the correct and the incorrect results, and gives a deeper understanding of the toxic mechanisms. The model has been applied to many compounds and the results are compared with experimental results reported for the corresponding Salmonella tests.

Organization and regulation of meta cleavage pathway genes for toluene and o-xylene derivative degradation in Pseudomonas stutzeri OX1.

Pseudomonas stutzeri OX1 meta pathway genes for toluene and o-xylene catabolism were analyzed, and loci encoding phenol hydroxylase, catechol 2,3-dioxygenase, 2-hydroxymuconate semialdehyde dehydrogenase, and 2-hydroxymuconate semialdehyde hydrolase were mapped. Phenol hydroxylase converted a broad range of substrates, as it was also able to transform the nongrowth substrates 2,4-dimethylphenol and 2,5-dimethylphenol into 3,5-dimethylcatechol and 3,6-dimethylcatechol, respectively, which, however, were not cleaved by catechol 2,3-dioxygenase. The identified gene cluster displayed a gene order similar to that of the Pseudomonas sp. strain CF600 dmp operon for phenol catabolism and was found to be coregulated by the tou operon activator TouR. A hypothesis about the evolution of the toluene and o-xylene catabolic pathway in P. stutzeri OX1 is discussed.

Development of biocatalysts carrying naphthalene dioxygenase and dihydrodiol dehydrogenase genes inducible in aerobic and anaerobic conditions.

We developed biocatalysts carrying naphthalene dioxygenase and dihydrodiol dehydrogenase genes cloned from plasmid pN3 of Pseudomonas fluoresceins N3 involved in naphthalene degradation, as an alternative approach to the production of hydroxylated compounds by chemical synthesis. Naphthalene dioxygenase is responsible for hydroxylation of the hydrocarbon into the corresponding 1,2-dihydro-1,2-dihydroxy derivative and dihydrodiol dehydrogenase is involved in the subsequent transformation into the 1,2-dihydroxy derivative. The first reaction strictly requires the presence of oxygen, essential for the dioxygenation reaction, while the second one can also be performed in anaerobic conditions that are optimal to avoid the easy oxidation of bioconversion products. Consequently, we constructed biocatalysts carrying the genes responsible for the biotransformation of hydrocarbons, inducible under aerobic and anaerobic conditions. We cloned the dioxygenase gene under its promoter, inducible by salicylic acid and the dihydrodiol dehydrogenase under the Pnar promoter of Escherichia coli, inducible by nitrate, in a nitrogen atmosphere, in order to develop biological systems with the possibility of controlling the expression of the cloned genes by the shift from aerobic to anaerobic conditions. Bioconversion experiments performed in aerobic conditions showed dihydrodiol production and dehydrogenase repression; as soon as cultures were switched to nitrogen, dihydrodiol dehydrogenation with an efficient production of 1,2-dihydroxyderivatives was observed.

Reaction centre accessibility. I. Calculation of reaction centre congestion and influence of structure flexibility

The fate of a reaction depends on many factors like the electronic reactivity of the ground state molecules, conformation/configuration needs and solvent influence. It is often impossible to predict with any certainty the result, in terms of yield and products, of the interaction between two reactants. Thus the role of reaction centre accessibility is definitely determining but is itself difficult to determine. Nevertheless, within the framework of reaction product prediction it is essential to search for an acceptable solution of predictive modelling. A new approach to both the calculation of steric congestion near reaction centres and its regulation is presented as a first step towards the prediction of centre accessibility. The approach is based on a two dimensional representation of molecules and on the calculation of the steric congestion of each branch, calculated by means of intersecting circles that indicate the extent of the reactivity space and the congestion space.

Reaction centre accessibility. II. Role of reaction centre congestion in the calculation of reaction centre accessibility

Accessibility to reaction centres is as important as electronic reactivity in determining the success of a reaction. The possibility of its calculation becomes a necessary requisite in the prediction of reaction products. Using a recently proposed approach to the calculation of reaction centre congestion based on a two dimensional representation of molecules, a new system has been realised that can quickly evaluate the desired accessibility. The system is based on the simulation of the steric interaction between reactants in different orientations. The calculation of an interaction energy for each orientation and their combination permits the approximate estimation of the reaction probability for the steric factors concerned. Even though all the operations were performed using a two-dimensional representation the results are encouraging. It is obvious that at this level it is impossible to predict face accessibility preference.

Prediction of organic reaction products: determining the best reaction conditions

We describe some of our most recent achievements concerning the selection of the best set of reaction conditions for a specific reaction. In particular, we will concentrate on the selection of the best solvent to minimize side reactions. The solvent should favor a kinetically controlled reaction if it is a good solvent for the transition state and a bad solvent for the ground state, decreasing the activation energy. Consequently, we need the descriptions of the transition state and of the state solvation. We generally apply the principle of "similarity in solvation"; i.e., we calculate an approximate similarity between reactants, transition states, and solvent molecules. The more the former are similar to the latter, the more they are solvated. This permits the selection and the ordering of the solvents. We considered other aspects of reaction conditions that will be briefly commented on. Some results will be presented to illustrate the power of the method.

A new biocatalyst for production of optically pure aryl epoxides by styrene monooxygenase from Pseudomonas fluorescens ST.

We developed a biocatalyst by cloning the styrene monooxygenase genes (styA and styB) from Pseudomonas fluorescens ST responsible for the oxidation of styrene to its corresponding epoxide. Recombinant Escherichia coli was able to oxidize different aryl vinyl and aryl ethenyl compounds to their corresponding optically pure epoxides. The results of bioconversions indicate the broad substrate preference of styrene monooxygenase and its potential for the production of several fine chemicals.

Specificity of substrate recognition by Pseudomonas fluorescens N3 dioxygenase. The role of the oxidation potential and molecular geometry.

Pseudomonas fluorescens N3 is able to grow on naphthalene as the sole carbon and energy source. The mutant TTC1, blocked at the dihydrodiol dehydrogenase level, which can transform the hydrocarbon into the corresponding dihydrodiol, has been used to produce bioconversion products. To rationalize the different grades of conversion obtained with different substrates, a study was performed using non-naphthalene derivatives, including benzenes, conjugated benzenes, and polycyclic aromatic hydrocarbons. The corresponding diols obtained by bioconversion have been isolated and characterized. A theoretical model that considers both energy and geometry factors has been proposed to rationalize the experimental data. Good agreement has been found between the calculated values and the experimental results.

Production of substituted naphthalene dihydrodiols by engineered Escherichia coli containing the cloned naphthalene 1,2-dioxygenase gene from Pseudomonas fluorescens N3.

Naphthalene dioxygenase, a key enzyme in the dihydroxylation of naphthalene, is encoded by the plasmid pN3, responsible for naphthalene metabolism in Pseudomonas fluorescens N3. The naphthalene dioxygenase, including all the sequences for its expression and the regulatory region, has been localized on the 4.3-kb HindIII-ClaI fragment and on the 3.5-kb HindIII fragment of the plasmid pN3, by Southern analysis using as probes nahA and nahR genes, the homologous genes of the plasmid NAH7 from Pseudomonas putida G7. We cloned in Escherichia coli JM109 the dioxygenase gene and its regulatory region and developed an efficient bacterial system inducible by salicylic acid, able to produce dihydrodiols. E. coli containing recombinant plasmids carrying the dioxygenase gene were analysed for their potential as a biocatalytic tool to produce dihydrodiols from different naphthalenes with the substituent on the aromatic ring at the alpha or beta position. The dihydrodiols, identified by HPLC (high-performance liquid chromatography) and 1H-NMR (nuclear magnetic resonance) were produced with yields ranging from 50 to 94%. The degree of bioconversion efficiency depends on the nature and the position of the substituent and indicates the broad substrate specificity of this dioxygenase and its potential for the production of a wide variety of fine chemicals.

Analysis of a theoretical model based on similarity for studying RNA base pairings.

A theoretical model for studying RNA base pairing is presented, based on similarity measures of the bases and corresponding indexes. Similarity calculations are made by evaluating atomic importance in molecules, a method that uses an original method for the calculation of electronic energy. The application of the model to both Watson-Crick and non-Watson-Crick pairings is commented on. Some theoretical considerations concerning the capability of the genetic code to repair dangerous mutations contribute to the ongoing debate.