Structural and functional characterization of two-domain laccase from Streptomyces viridochromogenes.

Laccase (EC 1.10.3.2) is one of the most common copper-containing oxidases found in many organisms and catalyses oxidation of primarily phenolic compounds by oxygen. A recently found bacterial laccase whose molecule is formed by two domains - the so called two-domain laccase (2DLac) or small laccase - has unusual resistance to inhibitors and an alkaline optimum of activity. The causes of these properties, as well as the biological function of two-domain laccases, are poorly understood. We performed an enzymatic and structural characterization of 2DLac from Streptomyces viridochromogenes (SvSL). It was cloned and overproduced in Escherichia coli. Phenolic compounds were oxidized in the presence of the enzyme under alkaline but not acidic conditions. Conversely, nonphenolic compounds were oxidized at acidic but not alkaline pH. SvSL catalysed oxidation of nonphenolic compounds more efficiently than that of phenols. Moreover, this two-domain laccase displayed a cytochrome c oxidase activity and exhibited no ferroxidase activity. The enzyme was resistant to specific inhibitors of copper-containing oxidases, such as NaN3 and NaF. We succeeded in generating X-ray quality crystals and solved their structure to a resolution of 2.4 Å. SvSL is a homotrimer in its native state. Comparison of its structure with that of a three-domain laccase revealed differences in the second coordination sphere of the T2/T3 centre and solvent channels. The role of these differences in the resistance of the enzyme to inhibitors and the activity at alkaline pH is under discussion.

New approaches to identification and activity estimation of glyphosate degradation enzymes.

We propose a new set of approaches, which allow identifying the primary enzymes of glyphosate (N-phosphonomethyl-glycine) attack, measuring their activities, and quantitative analysis of glyphosate degradation in vivo and in vitro. Using the developed approach we show that glyphosate degradation can follow different pathways depending on physiological characteristics of metabolizing strains: in Ochrobactrum anthropi GPK3 the initial cleavage reaction is catalyzed by glyphosate-oxidoreductase with the formation of aminomethylphosphonic acid and glyoxylate, whereas Achromobacter sp. MPS12 utilize C-P lyase, forming sarcosine. The proposed methodology has several advantages as compared to others described in the literature.

Oxidase reaction of the hybrid Mn-peroxidase of the fungus Panus tigrinus 8/18.

The hybrid Mn-peroxidase of the fungus Panus tigrinus 8/18 oxidized NADH in the absence of hydrogen peroxide, this being accompanied by the consumption of oxygen. The reaction of NADH oxidation started after a period of induction and completely depended on the presence of Mn(II). The reaction was inhibited in the presence of catalase and superoxide dismutase. Oxidation of NADH by the enzyme or by manganese(III)acetate was accompanied by the production of hydrogen peroxide and superoxide radicals. In the presence of NADH, the enzyme was transformed into a catalytically inactive oxidized form (compound III), and the latter was inactivated with bleaching of the heme. The substrate of the hybrid Mn-peroxidase (Mn(II)) reduced compound III to yield the native form of the enzyme and prevented its inactivation. It is assumed that the hybrid Mn-peroxidase used the formed hydrogen peroxide in the usual peroxidase reaction to produce Mn(III), which was involved in the formation of hydrogen peroxide and thus accelerated the peroxidase reaction. The reaction of NADH oxidation is a peroxidase reaction and the consumption of oxygen is due to its interaction with the products of NADH oxidation. The role of Mn(II) in the oxidation of NADH consisted in the production of hydrogen peroxide and the protection of the enzyme from inactivation.

Hybrid Mn-peroxidase from the ligninolytic fungus Panus tigrinus 8/18. Isolation, substrate specificity, and catalytic cycle.

Increased manganese concentration during submerged cultivation of the ligninolytic white rot fungus Panus tigrinus 8/18 on N-limited mineral medium resulted in the induction of Mn-peroxidase and laccase. The Mn-peroxidase was purified with the purity factor RZ (A(406)/A(280)) = 4.3. The purified enzyme catalyzed H2O2-dependent oxidation of phenol oxidase substrates (aromatic amines, 2,2;-azinobis-(3-ethylbenzthiazolinesulfonic acid), hydroquinone, 2,6-dimethoxyphenol) without Mn2+, which is not typical for the usual Mn-peroxidases. Guaiacol and 2,4,6-trichlorophenol were not oxidized in the absence of Mn2+. Study of absorption spectra of the intermediates of the catalytic cycle revealed that this peroxidase is able to complete the redox cycle, reducing one-electron oxidized intermediate (Compound II) by Mn2+, as well as by an organic substrate (hydroquinone). This means that the enzyme is a "hybrid" Mn-peroxidase, different from the common Mn-peroxidases from ligninolytic fungi.

Adaptation of the white-rot basidiomycete Panus tigrinus for transformation of high concentrations of chlorophenols.

During feed-batch cultivation of the white-rot fungus Panus tigrinus in a 5-l bioreactor on N-limited medium, 100, 200, 500, 1,000 and 2,000 mg 2,4,6-trichlorophenol (2,4,6-TCP) l(-1) were added sequentially after 90% removal of the previous portion of the toxicant. The addition of 500 mg 2,4,6-TCP l(-1) without preliminary adaptation killed the culture. The addition of 300 mg 2,4,6-TCP l(-1) without prior adaptation resulted in its slower removal than removal of 2,000 mg 2,4,6-TCP l(-1) by this adapted culture. After adaptation of P. tigrinus to 2,4,6-TCP in a 72-l bioreactor, the mixture of 2,4-dichlorophenol, 2,4,6-TCP, and pentachlorophenol, each at 500 mg x l(-1), was totally removed over 3 weeks. No lignin peroxidase activity was found in the course of cultivation of the fungus. Laccase activity was suppressed by addition of 2,4,6-TCP. Mn-peroxidase was found to be responsible for transformation of the chlorophenols. As final products of the process, several newly formed aromatic polymers, both chlorinated and non-chlorinated, were found in the culture liquid.

Transformation of 2,4,6-trichlorophenol by free and immobilized fungal laccase.

Laccase from the white rot fungus Coriolus versicolor was immobilized on Celite R-637 by covalent binding with glutaraldehyde. After a sharp primary decline in activity (up to 50%), the retained enzyme activity was stable over a storage period of 33 days at 4 degrees C. A comparative study of soluble and immobilized laccases revealed the increased resistance of immobilized enzyme to the unfavourable effects of alkaline pH, high temperature and the action of inhibitors. A combination of these properties of immobilized laccase resulted in the ability to oxidize 2,4,6-trichlorophenol (2,4,6-TCP) at 50 degrees C at pH 7.0. The reactions of soluble and immobilized laccase with 2,4,6-TCP were examined in the presence and absence of redox mediators. 3,5-Dichlorocatechol, 2,6-dichloro-1,4-benzoquinone and 2,6-dichloro-1,4-hydroquinone were found to be the primary products of 2,4,6-TCP oxidation by laccase; oligo- and polymeric compounds were also found.

Transformation of 2,4,6-trichlorophenol by the white rot fungi Panus tigrinus and Coriolus versicolor.

The toxicity of thirteen isomers of mono-, di-, tri- and pentachlorophenols was tested in potato-dextrose agar cultures of the white rot fungi Panus tigrinus and Coriolus versicolor. 2,4,6-Trichlorophenol (2,4,6-TCP) was chosen for further study of its toxicity and transformation in liquid cultures of these fungi. Two schemes of 2,4,6-TCP addition were tested to minimize its toxic effect to fungal cultures: stepwise addition from the moment of inoculation and single addition after five days of growth. In both cases the ligninolytic enzyme systems of both fungi were found to be responsible for 2,4,6-TCP transformation. 2,6-Dichloro-1,4-hydroquinol and 2,6-dichloro-1,4-benzoquinone were found as products of primary oxidation of 2,4,6-TCP by intact fungal cultures and purified ligninolytic enzymes, Mn-peroxidases and laccases of both fungi. However, primary attack of 2,4,6-TCP in P. tigrinus culture was conducted mainly by Mn-peroxidase, while in C. versicolor it was catalyzed predominantly by laccase, suggesting a different mode of regulation of these enzymes in the two fungi.

Reactions of blue and yellow fungal laccases with lignin model compounds.

Laccases of white-rot fungi Panus tigrinus, Phlebia radiata, and Phlebia tremellosa were isolated from cultures grown in liquid media which did not contain lignin and from the cultures grown on wheat straw. The physical and chemical properties of the laccases grown in submerged cultures were typical for blue fungal laccases. The laccases of the same fungi isolated from the solid-state cultures differed from the blue forms by lack of an absorption maximum at 610 nm. The typical blue laccases of P. tigrinus, Ph. radiata, and Ph. tremellosa acquired an ability to oxidize veratryl alcohol and a non-phenolic dimeric lignin model compound of beta-1-type only in the presence of a redox mediator, 2, 2'-azinobis(3-ethylbenzthiazolinesulfonic acid). The P. tigrinus and Ph. radiata yellow laccases catalyzed the oxidation of the same substrates without any mediator. The rate of the reaction of the blue laccases with a phenolic dimeric lignin model compound of beta-O-4-type was higher than that of the yellow laccases. The yellow laccases are apparently formed by the reaction of the blue laccases with low-molecular-weight lignin decomposition products.

Blue and yellow laccases of ligninolytic fungi.

Extracellular laccases from submerged cultures of Coriolus versicolor BKM F-116, Panus tigrinus 8/18, Phlebia radiata 79 (ATCC 64658), Phlebia tremellosa 77-51 and from cultures of Pa. tigrinus 8/18, Ph. radiata 79 and Agaricus bisporus D-649 grown on wheat straw (solid-state fermentation) were purified. All enzymes from submerged cultures had a blue colour and characteristic absorption and EPR spectra. Laccases from the solid-state cultures were yellow-brown and had no typical blue oxidase spectra and also showed atypical EPR spectra. Comparison of N-terminal amino acid sequences of purified laccases showed high homology between blue and yellow-brown laccase forms. Formation of yellow laccases as a result of binding of lignin-derived molecules by enzyme protein is proposed.