Glyphosate-Induced Phosphonatase Operons in Soil Bacteria of the Genus Achromobacter.

Achromobacter insolitus and Achromobacter aegrifaciens, bacterial degraders of the herbicide glyphosate, were found to induce phosphonatase (phosphonoacetaldehyde hydrolase, EC 3.11.1.1) when grown on minimal media with glyphosate as the sole source of phosphorus. The phosphonatases of the strains were purified to an electrophoretically homogeneous state and characterized. The enzymes differed in their kinetic characteristics and some other parameters from the previously described phosphonatases. The phosphonatase of A. insolitus was first revealed to separate into two stable forms, which had similar kinetic characteristics but interacted differently with affinity and ion-exchange resins. The genomes of the investigated bacteria were sequenced. The phosphonatase genes were identified, and their context was determined: the bacteria were shown to have gene clusters, which, besides the phosphonatase operon, included genes for LysR-type transcription activator (substrate sensor) and putative iron-containing oxygenase PhnHD homologous to monooxygenases PhnY and TmpB of marine organophosphonate degraders. Genes of 2-aminoethylphosphonate aminotransferase (PhnW, EC 2.6.1.37) were absent in the achromobacterial phosphonatase operons; instead, we revealed the presence of genes encoding the putative flavin oxidase HpnW. In silico simulation showed 1-hydroxy-2-aminoethylphosphonate to be the most likely substrate of the new monooxygenase, and a number of glycine derivatives structurally similar to glyphosate to be substrates of flavin oxidase.

A Novel Two-Domain Laccase with Middle Redox Potential: Physicochemical and Structural Properties.

The gene for a previously unexplored two-domain laccase was identified in the genome of actinobacterium Streptomyces carpinensis VKM Ac-1300. The two-domain laccase, named ScaSL, was produced in a heterologous expression system (Escherichia coli strain M15 [pREP4]). The enzyme was purified to homogeneity using affinity chromatography. ScaSL laccase, like most two-domain laccases, exhibited activity in the homotrimer form. However, unlike the most two-domain laccases, it was also active in multimeric forms. The enzyme exhibited maximum activity at 80°C and was thermally stable. Half-inactivation time of ScaSL at 80°C was 40 min. The laccase was able to oxidize a non-phenolic organic compound ABTS at a maximum rate at pH 4.7, and to oxidized a phenolic compound 2,6-dimethoxyphenol at a maximum rate at pH 7.5. The laccase stability was observed in the pH range 9-11. At pH 7.5, laccase was slightly inhibited by sodium azide, sodium fluoride, and sodium chloride; at pH 4.5, the laccase was completely inhibited by 100 mM sodium azide. The determined Km and kcat of the enzyme for ABTS were 0.1 mM and 20 s-1, respectively. The Km and kcat for 2,6-dimethoxyphenol were 0.84 mM and 0.36 s-1, respectively. ScaSL catalyzed polymerization of humic acids and lignin. Redox potential of the laccase was 0.472 ± 0.007 V. Thus, the ScaSL laccase is the first characterized two-domain laccase with a middle redox potential. Crystal structure of ScaSL was determined with 2.35 Å resolution. Comparative analysis of the structures of ScaSL and other two-domain laccases suggested that the middle potential of ScaSL may be associated with conformational differences in the position of the side groups of amino acids at position 230 (in ScaSL numbering), which belong to the second coordination sphere of the copper atom of the T1 center.

Multifunctional Enzyme with Endoglucanase and Alginase/Glucuronan Lyase Activities from Bacterium Cellulophaga lytica.

Cellulophaga lytica is a Gram-negative aerobic bacterium in the genome of which there are many genes encoding polysaccharide degrading enzymes. One of the enzymes named ClGP contains a glycoside hydrolase domain from the GH5 family and a polysaccharide lyase domain from the PL31 family. The enzyme also contains the TAT signaling peptide and the TIGR04183 domain that indicates extracellular nature of the enzyme. Phylogenetic analysis has shown that the enzymes most closely related to ClGP and containing all four domains (TAT, GH5, PL31, TIGR04183) are widespread among bacterial species belonging to the Flavobacteriaceae family. ClGP produced by the recombinant strain of E. coli was purified and characterized. ClGP exhibited activity of endoglucanase (EC 3.2.1.4) and catalyzed hydrolysis of ß-D-glucan, carboxymethyl cellulose sodium salt (CMC-Na), and amorphous cellulose, but failed to hydrolyze microcrystalline cellulose and xylan. Products of CMC hydrolysis were cellobiose and cellotriose, whereas ß-D-glucan was hydrolyzed to glucose, cellobiose, cellotetraose, and cellopentaose. ClGP was more active against the poly-ß-D-mannuronate blocks than against the poly-α-L-glucuronate blocks of alginic acid. This indicates that the enzyme is a polyM lyase (EC 4.2.2.3). ClGP was active against polyglucuronic acid, so it displayed a glucuronan lyase (EC 4.2.2.14) activity. The enzyme had a neutral pH-optimum, was stable in the pH range 6.0-8.0, and displayed moderate thermal stability. ClGP effectively saccharified two species of brown algae, Saccharina latissima and Laminaria digitata, that suggests its potential for use in the production of biofuel from macroalgae.

Metagenomes of Lichens Solorina crocea and Peltigera canina.

Lichen genomes are usually considered genomes of separately cultured mycobiont and photobiont. Analysis of lichen metagenomes can give important information on specific lichen-associated microorganisms that can affect lichen metabolism. Here, we report a metagenome of peltigeralean lichens, containing cyanobacterial (Peltigera canina) and cyanobacterial/green algal (Solorina crocea) partners.

Transformation of low molecular compounds and soil humic acid by two domain laccase of Streptomyces puniceus in the presence of ferulic and caffeic acids.

The two-domain bacterial laccases oxidize substrates at alkaline pH. The role of natural phenolic compounds in the oxidation of substrates by the enzyme is poorly understood. We have studied the role of ferulic and caffeic acids in the transformation of low molecular weight substrates and of soil humic acid (HA) by two-domain laccase of Streptomyces puniceus (SpSL, previously undescribed). A gene encoding a two-domain laccase was cloned from S. puniceus and over-expressed in Escherichia coli. The recombinant protein was purified by affinity chromatography to an electrophoretically homogeneous state. The enzyme showed high thermal stability, alkaline pH optimum for the oxidation of phenolic substrates and an acidic pH optimum for the oxidation of K4[Fe(CN)6] (potassium ferrocyanide) and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt). Phenolic compounds were oxidized with lower efficiency than K4[Fe(CN)6] and ABTS. The SpSL did not oxidize 3.4-dimethoxybenzoic alcohol and p-hydroxybenzoic acid neither in the absence of phenolic acids nor in their presence. The enzyme polymerized HA-the amount of its high molecular weight fraction (>80 kDa) increased at the expense of low MW fraction (10 kDa). The addition of phenolic acids as potential mediators did not cause the destruction of HA by SpSL. In the absence of the HA, the enzyme polymerized caffeic and ferulic acids to macromolecular fractions (>80 kDa and 10-12 kDa). The interaction of SpSL with HA in the presence of phenolic acids caused an increase in the amount of HA high MW fraction and a two-fold increase in the molecular weight of its low MW fraction (from 10 to 20 kDa), suggesting a cross-coupling reaction. Infrared and solution-state 1H-NMR spectroscopy revealed an increase in the aromaticity of HA after its interaction with phenolic acids. The results of the study expand our knowledge on the transformation of natural substrates by two-domain bacterial laccases and indicate a potentially important role of the enzyme in the formation of soil organic matter (SOM) at alkaline pH values.

A 2,5-Dihydroxybenzoic Acid-Gelatin Conjugate Inhibits the Basal and Hsp90-Stimulated Migration and Invasion of Tumor Cells.

The extracellular cell surface-associated and soluble heat shock protein 90 (Hsp90) is known to participate in the migration and invasion of tumor cells. Earlier, we demonstrated that plasma membrane-associated heparan sulfate proteoglycans (HSPGs) bind the extracellular Hsp90 and thereby promote the Hsp90-mediated motility of tumor cells. Here, we showed that a conjugate of 2,5-dihydroxybenzoic acid with gelatin (2,5-DHBA-gelatin), a synthetic polymer with heparin-like properties, suppressed the basal (unstimulated) migration and invasion of human glioblastoma A-172 and fibrosarcoma HT1080 cells, which was accompanied by the detachment of a fraction of Hsp90 from cell surface HSPGs. The polymeric conjugate also inhibited the migration/invasion of cells stimulated by exogenous soluble native Hsp90, which correlated with the inhibition of the attachment of soluble Hsp90 to cell surface HSPGs. The action of the 2,5-DHBA-gelatin conjugate on the motility of A-172 and HT1080 cells was similar to that of heparin. The results demonstrate a potential of the 2,5-DHBA-gelatin polymer for the development of antimetastatic drugs targeting cell motility and a possible role of extracellular Hsp90 in the suppression of the migration and invasion of tumor cells mediated by the 2,5-DHBA-gelatin conjugate and heparin.

Draft Genome Sequence of Glyphosate-Degrading Achromobacter insolitus Strain Kg 19 (VKM B-3295), Isolated from Agricultural Soil.

The genome of an Achromobacter insolitus strain isolated from an agricultural soil polluted with the herbicide glyphosate is reported. The genome size is 6.4 Mb, with an average G+C content of 65.2%. These genomic data could contribute to a better understanding of the biochemistry and regulatory mechanisms of the microbial degradation of glyphosate and aminomethylphosphonate.

Transformation of cellobiose during the interaction of cellobiose dehydrogenase and ß-glucosidase of Cerrena unicolor.

This work investigated the regulatory role of the interaction between cellobiose dehydrogenase (CDH) and ß-glucosidase (ß-GLU) in the conversion of cellobiose into cellobionolactone or glucose in vitro. To study the regulation, the two enzymes were isolated from the culture medium of the fungus Cerrena unicolor grown on a medium with microcrystalline cellulose. The enzymes were obtained in an electrophoretically homogeneous state. Their properties were studied. Both enzymes had acidic pH optima and were more stable in the acidic pH range. CDH was moderately thermostable, while ß-GLU had a low thermostability. Both enzymes efficiently catalyzed the transformation of cellobiose. A mixture of CDH and ß-GLU transformed cellobiose to glucose or cellobionolactone in the presence of various concentrations of laccase and hydroquinone. Formation of glucose and cellobionolactone in vitro during the competition between CDH and ß-GLU for cellobiose depended on the availability of quinones, formed as a result of the interaction of laccase and hydroquinone, for CDH. At low laccase and hydroquinone concentrations, the formation of glucose was found to predominate over that of cellobionolactone. The possible physiological role of the enzymes' interaction is discussed.

Organophosphonates utilization by soil strains of Ochrobactrum anthropi and Achromobacter sp.

Four bacterial strains from glyphosate- or alkylphosphonates-contaminated soils were tested for ability to utilize different organophosphonates. All studied strains readily utilized methylphosphonic acid and a number of other phosphonates, but differed in their ability to degrade glyphosate. Only strains Ochrobactrum anthropi GPK 3 and Achromobacter sp. Kg 16 utilized this compound after isolation from enrichment cultures with glyphosate. Achromobacter sp. MPK 7 from the same enrichment culture, similar to Achromobacter sp. MPS 12 from methylphosphonate-polluted source, required adaptation to growth on GP. Studied strains varied significantly in their growth parameters, efficiency of phosphonates degradation and characteristic products of this process, as well as in their energy metabolism. These differences give grounds to propose a possible model of interaction between these strains in microbial consortium in phosphonate-contaminated soils.

Xylanases of Cellulomonas flavigena: expression, biochemical characterization, and biotechnological potential.

Four xylanases of Cellulomonas flavigena were cloned, expressed in Escherichia coli and purified. Three enzymes (CFXyl1, CFXyl2, and CFXyl4) were from the GH10 family, while CFXyl3 was from the GH11 family. The enzymes possessed moderate temperature stability and a neutral pH optimum. The enzymes were more stable at alkaline pH values. CFXyl1 and CFXyl2 hydrolyzed xylan to form xylobiose, xylotriose, xylohexaose, xylopentaose, and xylose, which is typical for GH10. CFXyl3 (GH11) and CFXyl4 (GH10) formed the same xylooligosaccharides, but xylose was formed in small amounts. The xylanases made efficient saccharification of rye, wheat and oat, common components of animal feed, which indicates their high biotechnological potential.

Glyphosate acetylation as a specific trait of Achromobacter sp. Kg 16 physiology.

The growth parameters of Achromobacter sp. Kg 16 (VKM B-2534 D), such as biomass and maximum specific growth rate, depended only on the source of phosphorus in the medium, but not on the carbon source or the presence of growth factors. With glyphosate as a sole phosphorus source, they were still 40-50 % lower than in media supplemented with orthophosphate or other organophosphonate-methylphosphonic acid. At the first time process of glyphosate acetylation and accumulation of acetylglyphosate in culture medium were revealed in this strain. Acetylglyphosate isolated from cultural liquid was identified by mass spectroscopy; its mass spectrum fully corresponded with that of chemically synthesized acetylglyphosate. Even poorer growth was observed in media with acetylglyphosate: although the strain was able to utilize this compound as a sole source of phosphorus, the maximum biomass was still 58-70 % lower than with glyphosate. The presence of acetylglyphosate in culture medium could also hinder the utilization of glyphosate as a phosphorus source. Therefore, the acetylation of glyphosate may be a specific feature of Achromobacter sp. Kg 16 responsible for its poor growth on this compound.

Recombinant xylanase from Streptomyces coelicolor Ac-738: characterization and the effect on xylan-containing products.

A xylanase gene was isolated from the genomic DNA of Streptomyces coelicolor Ac-738. The 723-bp full-length gene encoded a 241-amino acid peptide consisting of a 49-residue putative TAT signal peptide and a glycoside hydrolase family-11 domain. The mature enzyme called XSC738 was expressed in Escherichia coli M15[pREP4]. The electrophoretically homogeneous protein with a specific activity of 167 U/mg for beechwood xylan was purified. The pH optimum of XSC738 was at pH 6; a high activity was retained within a pH range of 4.5-8.5. The enzyme was thermostable at 50-60 °C and retained an activity at pH 3.0-7.0. Xylanase XSC738 was activated by Mn²âº, Co²âº and largely inhibited by Cd²âº, SDS and EDTA. The products of xylan hydrolysis were mainly xylobiose, xylotriose, xylopentaose and xylohexose. Xylotetraose appeared as a minor product. Processing of such agricultural xylan-containing products as wheat, oats, soy flour and wheat bran by xylanase resulted in an increased content of sugars.

Distribution of glyphosate and methylphosphonate catabolism systems in soil bacteria Ochrobactrum anthropi and Achromobacter sp.

Bacterial strains capable of utilizing methylphosphonic acid (MP) or glyphosate (GP) as the sole sources of phosphorus were isolated from soils contaminated with these organophosphonates. The strains isolated from MP-contaminated soils grew on MP and failed to grow on GP. One group of the isolates from GP-contaminated soils grew only on MP, while the other one grew on MP and GP. Strains Achromobacter sp. MPS 12 (VKM B-2694), MP degraders group, and Ochrobactrum anthropi GPK 3 (VKM B-2554D), GP degraders group, demonstrated the best degradative capabilities towards MP and GP, respectively, and were studied for the distribution of their organophosphonate catabolism systems. In Achromobacter sp. MPS 12, degradation of MP was catalyzed by C-P lyase incapable of degrading GP (C-P lyase I). Adaptation to growth on GP yielded the strain Achromobacter sp. MPS 12A, which retained its ability to degrade MP via C-P lyase I and was capable of degrading GP with formation of sarcosine, thus suggesting the involvement of a GP-specific C-P lyase II. O. anthropi GPK 3 also degraded MP via C-P lyase I, but degradation of GP in it was initiated by glyphosate oxidoreductase, which was followed by product transformation via the phosphonatase pathway.

Purification, crystallization and preliminary X-ray structure analysis of the laccase from Ganoderma lucidum.

The ligninolytic enzymes of the basidiomycetes play a key role in the global carbon cycle. A characteristic property of these enzymes is their broad substrate specificity, which has led to their use in various biotechnologies, thus stimulating research into the three-dimensional structures of ligninolytic enzymes. This paper presents the purification, crystallization and preliminary X-ray analysis of the laccase from the ligninolytic basidiomycete Ganoderma lucidum.

Bioremediation of glyphosate-contaminated soils.

Based on the results of laboratory and field experiments, we performed a comprehensive assessment of the bioremediation efficiency of glyphosate-contaminated soddy-podzol soil. The selected bacterial strains Achromobacter sp. Kg 16 (VKM B-2534D) and Ochrobactrum anthropi GPK 3 (VKM B-2554D) were used for the aerobic degradation of glyphosate. They demonstrated high viability in soil with the tenfold higher content of glyphosate than the recommended dose for the single in situ treatment of weeds. The strains provided a two- to threefold higher rate of glyphosate degradation as compared to indigenous soil microbial community. Within 1-2 weeks after the strain introduction, the glyphosate content of the treated soil decreased and integral toxicity and phytotoxicity diminished to values of non-contaminated soil. The decrease in the glyphosate content restored soil biological activity, as is evident from a more than twofold increase in the dehydrogenase activity of indigenous soil microorganisms and their biomass (1.2-fold and 1.6-fold for saprotrophic bacteria and fungi, respectively). The glyphosate-degrading strains used in this study are not pathogenic for mammals and do not exhibit integral toxicity and phytotoxicity. Therefore, these strains are suitable for the efficient, ecologically safe, and rapid bioremediation of glyphosate-contaminated soils.

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. RESULTS: The structure of a laccase from the white-rot fungus Lentinus (Panus) tigrinus, a glycoenzyme involved in lignin biodegradation, was solved at 1.5 A. It reveals a asymmetric unit containing two laccase molecules (A and B). The progressive reduction of the copper ions centers obtained by the long-term exposure of the crystals to the high-intensity X-ray synchrotron beam radiation under aerobic conditions and high pH allowed us to detect two sequential intermediates in the molecular oxygen reduction pathway: the "peroxide" and the "native" intermediates, previously hypothesized through spectroscopic, kinetic and molecular mechanics studies. Specifically the electron-density maps revealed the presence of an end-on bridging, micro-eta 1:eta 1 peroxide ion between the two T3 coppers in molecule B, result of a two-electrons reduction, whereas in molecule A an oxo ion bridging the three coppers of the T2/T3 cluster (micro3-oxo bridge) together with an hydroxide ion externally bridging the two T3 copper ions, products of the four-electrons reduction of molecular oxygen, were best modelled. CONCLUSION: This is the first structure of a multicopper oxidase which allowed the detection of two intermediates in the molecular oxygen reduction and splitting. The observed features allow to positively substantiate an accurate mechanism of dioxygen reduction catalyzed by multicopper oxidases providing general insights into the reductive cleavage of the O-O bonds, a leading problem in many areas of biology.

Laccases produced by lichens of the order Peltigerales.

'Large' and 'small' fractions of laccase were found in the thalli of lichens Solorina crocea and Peltigera aphthosa. In both lichens, 'large', possibly dimeric, laccases were determined as 175 and 165 kDa (based on the gel filtration data), and 'small' ones were 76 and 97 kDa (according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis data), respectively. By their substrate specificity, pH optima, and thermostability, they were typical laccases. The fractions of 'small' laccases of 45 kDa from S. crocea and 55 kDa from P. aphthosa consisted of two enzymes.

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, 0.2 M CaCl2, 100 mM Tris-HCl pH 7.5. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 54.2, b = 111.6, c = 97.1, beta = 97.7 degrees , and contain 46% solvent. A complete native data set was collected to 1.4 A resolution at the copper edge. Molecular replacement using the Coprinus cinereus laccase structure (PDB code 1hfu) as a starting model was performed and initial electron-density maps revealed the presence of a full complement of copper ions. Model refinement is in progress. The P. tigrinus laccase structural model exhibits the highest resolution available to date and will assist in further elucidation of the catalytic mechanism and electron-transfer processes for this class of enzymes.