The response of soil Arthrobacter agilis lush13 to changing conditions: Transition between vegetative and dormant state.

This work was aimed at studying the response of soil non-spore-forming actinobacterial strain Arthrobacter agilis Lush 13 to changing natural conditions, such as nutrient availability and the presence of degradable and recalcitrant aliphatic and aromatic substrates. The A. agilis strain Lush13 was able to degrade octane, nonane, hexadecane, benzoate, phenol, and 2,3-, 2,4-, 2,5-, 2,6-dichlorophenols, but not grew on 3,4-dichlorophenol, 2,3,4-, 2,4,5-, 2,4,6-trichlorophenol (TCP), pentachlorophenol (PCP), 2-chlorobenzoate, 3-chlorobenzoate, 3,5-dichlorobenzoate, 2,4-dichlorobenzoate.

Benzoate degradation by Rhodococcus opacus 1CP after dormancy: Characterization of dioxygenases involved in the process.

The process of benzoate degradation by strain Rhodococcus opacus 1CP after a five-year dormancy was investigated and its peculiarities were revealed. The strain was shown to be capable of growth on benzoate at a concentration of up to 10 g L(-1). The substrate specificity of benzoate dioxygenase (BDO) during the culture growth on a medium with a low (200-250 mg L(-1)) and high (4 g L(-1)) concentration of benzoate was assessed.

Investigating the Role of Conformational Effects on Laccase Stability and Hyperactivation under Stress Conditions.

Fungal laccase from Steccherinum ochraceum 1833 displays remarkable stability under different harsh conditions: organic/buffer mixtures, thermal treatment, and microwave radiation. The behavior is particularly significant in the light of the sharp inactivation observed for two different fungal laccases. Laccase from S.

Degradation of phenanthrene by the rhizobacterium Ensifer meliloti.

The biodegradation of the polycyclic aromatic hydrocarbon phenantherene by the rhizobacterial strain Ensifer meliloti P221, isolated from the root zone of plant grown in PAH-contaminated soil was studied. Bacterial growth and phenanthrene degradation under the influence of root-exuded organic acids were also investigated. Analysis of the metabolites produced by the strain by using thin-layer chromatography, gas chromatography, high-pressure liquid chromatography, and mass-spectrometry revealed that phenanthrene is bioconverted via two parallel pathways.

Comparison of the substrate specificity of two ring-hydroxylating dioxygenases from Sphingomonas sp. VKM B-2434 to polycyclic aromatic hydrocarbons.

The genes of two ring-hydroxylating dioxygenases (RHDs) of Sphingomonas sp. VKM B-2434 were cloned and expressed in Escherichia coli. The relative values of the RHD specificity constants were estimated for six polycyclic aromatic hydrocarbons (PAHs) based on the kinetics of PAH mixture conversion by the recombinant strains.

Structural basis for the substrate specificity and the absence of dehalogenation activity in 2-chloromuconate cycloisomerase from Rhodococcus opacus 1CP.

2-Chloromuconate cycloisomerase from the Gram-positive bacterium Rhodococcus opacus 1CP (Rho-2-CMCI) is an enzyme of a modified ortho-pathway, in which 2-chlorophenol is degraded using 3-chlorocatechol as the central intermediate. In general, the chloromuconate cycloisomerases catalyze not only the cycloisomerization, but also the process of dehalogenation of the chloromuconate to dienelactone. However Rho-2-CMCI, unlike the homologous enzymes from the Gram-negative bacteria, is very specific for only one position of the chloride on the substrate chloromuconate.

Chloromuconolactone dehalogenase ClcF of actinobacteria.

This work investigated the distribution of the clcF gene in actinobacteria isolated from different ecotopes. The gene encodes chloromuconolactone dehalogenase (CMLD) ClcF, the enzyme found to date in only one representative of Gram-positive bacteria, Rhodococcus opacus 1CP, adapted to 2-chlorophenol (2CP). Using primers specific to the clcF gene, from the DNA matrix of rhodococcal strains closely related to species Rhodococcus wratislaviensis (P1, P12, P13, P20, G10, KT112, KT723, BO1) we obtained PCR products whose nucleotide sequences were 100% identical to that of the clcF gene from strain R.

X-ray crystallographic and molecular docking studies on a unique chloromuconolactone dehalogenase from Rhodococcus opacus 1CP.

5-Chloromuconolactone dehalogenase (5-CMLD) is a unique enzyme that catalyzes the conversion of 5-chloromuconolactone into cis-dienelactone in the new modified ortho-pathway of the 3-chlorocatechol degradation by Rhodococcus opacus 1CP. In all other known chlorocatechol pathways the dehalogenation is a spontaneous secondary reaction of the unstable chloromuconate intermediate following the lactonization process catalyzed by the muconate cycloisomerases. The crystallographic structure of the decameric 5-CMLD was solved by Molecular Replacement, using the coordinates of the low resolution structure of the highly homologous muconolactone isomerase, an enzyme of the conventional ortho-pathway.

X-ray structures of 4-chlorocatechol 1,2-dioxygenase adducts with substituted catechols: new perspectives in the molecular basis of intradiol ring cleaving dioxygenases specificity.

The crystallographic structures of 4-chlorocatechol 1,2-dioxygenase (4-CCD) complexes with 3,5-dichlorocatechol, protocatechuate (3,4-dihydroxybenzoate), hydroxyquinol (benzen-1,2,4-triol) and pyrogallol (benzen-1,2,3-triol), which act as substrates or inhibitors of the enzyme, have been determined and analyzed. 4-CCD from the Gram-positive bacterium Rhodococcus opacus 1CP is a Fe(III) ion containing enzyme specialized in the aerobic biodegradation of chlorocatechols. The structures of the 4-CCD complexes show that the catechols bind the catalytic iron ion in a bidentate mode displacing Tyr169 and the benzoate ion (found in the native enzyme structure) from the metal coordination sphere, as found in other adducts of intradiol dioxygenases with substrates.

Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus.

The lignin-degrading, biopulping white-rot fungus Physisporinus rivulosus secretes several laccases of distinct features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. Here we describe the cloning, heterologous expression and structural and enzymatic characterisation of two previously undescribed P. rivulosus laccases.

Reaction intermediates and redox state changes in a blue laccase from Steccherinum ochraceum observed by crystallographic high/low X-ray dose experiments.

The crystal structure of a blue laccase from Steccherinum ochraceum has been solved at 2.0Å of resolution using a classic data acquisition from a single crystal. The overall structural features are typical of this class of enzymes, however, distances inside the trinuclear copper cluster are indicative of a reduction of the metal centers induced by free electrons produced during the X-ray data collection.

Improved xenobiotic-degrading activity of Rhodococcus opacus strain 1cp after dormancy.

The goals of the present work were as follows: to obtain the dormant forms of R. opacus 1cp; to study the phenotypic variability during their germination; to compare phenotypic variants during the growth on selective and elective media; and to reveal changes in the ability of the strain to destruct xenobiotics that had not been degradable before dormancy. It was shown that Rhodococcus opacus 1cp (the strain degrading chlorinated phenols) became able to utilize a broader spectrum of xenobiotics after storage in the dormant state.

Effect of alkyl hydroxybenzenes on the properties of dioxygenases.

The aim of the present work was to investigate the influence of alkylhydroxybenzenes (AHBs) and tyrosol, which belong to cell differentiation factors d(1) group of autoregulators on properties of biodegradation enzymes, catechol 1,2-dioxygenase (Cat 1,2-DO) and methylcatechol 1,2-dioxygenase (MCat 1,2-DO) of Rhodococcus opacus 6a. AHBs were found to have a greater effect on MCat 1,2-DO than on Cat 1,2-DO. It was expressed by more pronounced changes in the activity of MCat 1,2-DO with unsubstituted catechol at different AHB concentrations and by increasing thermostability of MCat 1,2-DO compared to Cat 1,2-DO under the protective action of AHBs.

Multisubstrate kinetics of PAH mixture biodegradation: analysis in the double-logarithmic plot.

The proposed method of kinetic analysis of aqueous-phase biodegradation of polycyclic aromatic hydrocarbons (PAH) mixture presupposes representation of kinetic curves for each pair of mixture components, S(x) and S(y), in double-logarithmic coordinates (ln S(x); ln S(y)). If PAH mixture conversion corresponds to the multisubstrate model with a common active site, then the graphs in double-logarithmic coordinates are straight lines with the angular coefficients equal to the ratio of respective first-order rate constants k(y)(x)= V(y)K(x)/K(y)V(x), where K(x) and K(y) are half-saturation constants, V(x) and V( y ) are the maximum conversion rates for substrates S(x) and S(y); the graph slope does not depend on any concentrations and remains constant during the change of reaction rates as a result of inhibition, induction/inactivation of enzymes or biomass growth. The formulated method has been used to analyze PAH mixture conversion by the culture of Sphingomonas sp.

Catechol 1,2-dioxygenase from the Gram-positive Rhodococcus opacus 1CP: quantitative structure/activity relationship and the crystal structures of native enzyme and catechols adducts.

The first crystallographic structures of a catechol 1,2-dioxygenase from a Gram-positive bacterium Rhodococcus opacus 1CP (Rho 1,2-CTD), a Fe(III) ion containing enzyme specialized in the aerobic biodegradation of catechols, and its adducts with catechol, 3-methylcatechol, 4-methylcatechol, pyrogallol (benzene-1,2,3-triol), 3-chlorocatechol, 4-chlorocatechol, 3,5-dichlorocatechol, 4,5-dichlorocatechol and protocatechuate (3,4-dihydroxybenzoate) have been determined and analyzed. This study represents the first extensive characterization of catechols adducts of 1,2-CTDs. The structural analyses reveal the diverse modes of binding to the active metal iron ion of the tested catechols thus allowing to identify the residues selectively involved in recognition of the diverse substrates by this class of enzymes.

Role of surfactants in optimizing fluorene assimilation and intermediate formation by Rhodococcus rhodochrous VKM B-2469.

Biodegradation of fluorene by Rhodococcus rhodochrous VKM B-2469 was investigated and optimized by adding non-ionic surfactants to the liquid media. The utilization of 1-1.5% Tween 60 or 1% Triton X100 allowed to solubilize 1 mM fluorene over 150 times more than in water medium (from 9-11 microM to above 1.

Variability of enzyme system of Nocardioform bacteria as a basis of their metabolic activity.

The present review describes some aspects of organization of biodegradative pathways of Nocardioform microorganisms, first of all, with respect to their ability to degrade aromatic compounds, mostly methylbenzoate, chlorosubstituted phenols, and chlorinated biphenyls and the intermediates of their transformation: 4-chlorobenzoate and para-hydroxybenzoate. Various enzyme systems induced during degradation processes are defined. The ability of microorganisms to induce a few key enzymes under the influence of xenobiotics is described.

Biotransformation of chloroaromatics: the impact of bioavailability and substrate specificity.

The effect of surfactants on the biodegradation of mono-aromatic hydrocarbons such as benzene, chlorobenzene and 1,2-dichlorobenzene by an Escherichia coli JMI09(MI) recombinant strain, carrying a gene cluster containing the genes for benzene dioxygenase, cis-benzene dihydrodiol dehydrogenase, and catechol 2,3-dioxygenase from Pseudomonas putida ML2, has been investigated. We observed that the efficiency of the benzene dioxygenase catalyzed conversions to cis-dihydrodiols depends on the balance among real substrate specificity, bioavailability, and toxicity effects of highly concentrated aromatic hydrocarbons. The utilization of non ionic surfactants makes it possible to partly overcome the limiting step of biodegradation processes for scarcely water soluble hydrocarbons hindered by their limited bioavailability.

Conversion of polycyclic aromatic hydrocarbons by Sphingomonas sp. VKM B-2434.

A versatile bacterial strain able to convert polycyclic aromatic hydrocarbons (PAHs) was isolated, and a conversion by the isolate of both individual substances and PAH mixtures was investigated. The strain belonged to the Sphingomonas genus as determined on the basis of 16S rRNA analysis and was designated as VKM B-2434. The strain used naphthalene, acenaphthene, phenanthrene, anthracene and fluoranthene as a sole source of carbon and energy, and cometabolically oxidized fluorene, pyrene, benz[a]anthracene, chrysene and benzo[a]pyrene.

Crystal structure of a blue laccase from Lentinus tigrinus: evidences for intermediates in the molecular oxygen reductive splitting by multicopper oxidases.

Background: Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in pathogenesis, immunogenesis and morphogenesis of organisms and in the metabolic turnover of complex organic substances. They catalyze the coupling between the four one-electron oxidations of a broad range of substrates with the four-electron reduction of dioxygen to water. These catalytic processes are made possible by the contemporaneous presence of at least four copper ion sites, classified according to their spectroscopic properties: one type 1 (T1) site where the electrons from the reducing substrates are accepted, one type 2 (T2), and a coupled binuclear type 3 pair (T3) which are assembled in a T2/T3 trinuclear cluster where the electrons are transferred to perform the O2 reduction to H2O.

Intradiol pathway of para-cresol conversion by Rhodococcus opacus 1CP.

The growth of Rhodococcus opacus 1CP in medium with different concentrations of p-cresol as the sole source of carbon and energy was studied. It was shown that the optimal concentration of p-cresol was 600 mg/L. The ability of this strain to transform practically all amounts of p-cresol to 4-methylcatechol followed by its utilization through ortho-pathway was shown.

Hydroxyquinol pathway for microbial degradation of halogenated aromatic compounds.

Several peripheral metabolic pathways can be used by microorganisms to degrade toxic aromatic compounds that are known to pollute the environment. Hydroxyquinol (1,2,4-trihydroxybenzene) is one of the central intermediates in the degradative pathway of a large variety of aromatic compounds. The present review describes the microorganisms involved in the degradative pathway, the key enzymes involved in the formation and splitting of the aromatic ring of (chloro)hydroxyquinol as well as the central intermediates formed.

Crystal structure of 3-chlorocatechol 1,2-dioxygenase key enzyme of a new modified ortho-pathway from the Gram-positive Rhodococcus opacus 1CP grown on 2-chlorophenol.

The crystal structure of the 3-chlorocatechol 1,2-dioxygenase from the Gram-positive bacterium Rhodococcus opacus (erythropolis) 1CP, a Fe(III) ion-containing enzyme specialized in the aerobic biodegradation of 3-chloro- and methyl-substituted catechols, has been solved by molecular replacement techniques using the coordinates of 4-chlorocatechol 1,2-dioxygenase from the same organism (PDB code 1S9A) as a starting model and refined at 1.9 A resolution (R(free) 21.9%; R-factor 17.

Crystallization and preliminary structure analysis of the blue laccase from the ligninolytic fungus Panus tigrinus.

The blue laccase from the white-rot basidiomycete fungus Panus tigrinus, an enzyme involved in lignin biodegradation, has been crystallized. P. tigrinus laccase crystals grew within one week at 296 K using the sitting-drop vapour-diffusion method in 22%(w/v) PEG 4000, 0.

Crystal structure of the hydroxyquinol 1,2-dioxygenase from Nocardioides simplex 3E, a key enzyme involved in polychlorinated aromatics biodegradation.

Hydroxyquinol 1,2-dioxygenase (1,2-HQD) catalyzes the ring cleavage of hydroxyquinol (1,2,4-trihydroxybenzene), a central intermediate in the degradation of aromatic compounds including a variety of particularly recalcitrant polychloro- and nitroaromatic pollutants. We report here the primary sequence determination and the analysis of the crystal structure of the 1,2-HQD from Nocardioides simplex 3E solved at 1.75 A resolution using the multiple wavelength anomalous dispersion of the two catalytic irons (1 Fe/293 amino acids).