Fungal Peroxidases Research Articles

Comparative evaluation of manganese peroxidase- and Mn(III)-initiated peroxidation of C18 unsaturated fatty acids by different methods

The peroxidation of C18 unsaturated fatty acids by fungal manganese peroxidase (MnP)/Mn(II) and by chelated Mn(III) was studied with application of three different methods: by monitoring oxygen consumption, by measuring conjugated dienes and by thiobarbituric acid-reactive substances (TBARS) formation. All tested polyunsaturated fatty acids (PUFAs) were oxidized by MnP in the presence of Mn(II) ions but the rate of their oxidation was not directly related to degree of their unsaturation. As it has been shown by monitoring oxygen consumption and conjugated dienes formation the linoleic acid was the most easily oxidizable fatty acid for MnP/Mn(II) and chelated Mn(III). However, when the lipid peroxidation (LPO) activity was monitored by TBARS formation the linolenic acid gave the highest results. High accumulation of TBARS was also recorded during peroxidation of linoleic acid initiated by MnP/Mn(II). Action of Mn(III)-tartrate on the PUFAs mimics action of MnP in the presence of Mn(II) indicating that Mn(III) ions are involved in LPO initiation. Although in our experiments Mn(III) tartrate gave faster than MnP/Mn(II) initial oxidation of the unsaturated fatty acids with consumption of O 2 and formation of conjugated dienes the process was not productive and did not support further development of LPO. The higher effectiveness of MnP/Mn(II)-initiated LPO system depends on the turnover of manganese provided by MnP. It is proposed that the oxygen consumption assay is the best express method for evaluation of MnP- and Mn(III)-initiated peroxidation of C18 unsaturated fatty acids.

Effects of flax lignin addition on enzymatic oxidation of poly(ethylene adipate) urethanes

Lignin use in polymer industry has recently become very attractive from both economical and environmental reasons. In the specific case of blending, the addition of low lignin amounts was found to improve the properties and extends the application field of other natural or synthetic polymers, but the effects of oxidative enzymes on resulting blends are widely unknown. Thereby, the current study was carried out to determine some potential effects of small flax lignin concentrations (4.2 and 9.3 wt%) on the enzymatic degradation of a poly(ethylene adipate) urethane (PU). Thin cast films of PU and its lignin blends were incubated for 3 days at 30 °C with buffered solutions of fungal peroxidase and laccase extracted from Aspergillus sp., and compared with the untreated ones. Changes in surface structure and morphology were investigated by attenuated total reflectance–Fourier transform infrared spectroscopy (ATR–FTIR) and scanning electron microscopy (SEM), while the impact on bulk was assessed from tensile tests and thermogravimetry analysis (TGA). Although the addition of flax lignin reduces the surface structural modifications after enzyme treatment, the morphology, tensile and thermo-oxidative characteristics are still affected, with laccase showing the higher degradative efficiency. The lignin concentration, its high impact on the resulting blends morphology and relative low resistance to laccase and peroxidase degradation was the most important factors proved to driven the enzymatic oxidation.

Sequencing the fungal tree of life

Sequencing the Fungal Tree of Life F. Martin 1 , D. Cullen 2 , D. Hibbett 3 , A. Pisabarro 4 , J. W. Spatafora 5 , S. E. Baker 6,7, I. V. Grigoriev 7 UMR INRA/UHP 1136, Interactions Arbres/Micro-Organismes, INRA-Nancy, 54280 Champenoux, France University of Wisconsin-Madison, Madison, WI, USA Clark University, Worcester, MA, USA Department of Agrarian Production, Public University of Navarre, 31006 Pamplona, Spain Dept. Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331 Chemical and Biological Process Development Group, Pacific Northwest National Laboratory, Richland, Washington, USA, 99352 US DOE Joint Genome Institute, Walnut Creek, California, USA March 2011 The work conducted by the U.S. Department of Energy Joint Genome Institute is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02- 05CH11231 DISCLAIMER This document was prepared as an account of work sponsored by the United States Government. While this document is believed to contain correct information, neither the United States Government nor any agency thereof, nor The Regents of the University of California, nor any of their employees, makes any warranty, express or implied, or assumes any legal responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by its trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof, or The Regents of the University of California. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof or The Regents of the University of California.

Oxidation of pharmaceutically active compounds by a ligninolytic fungal peroxidase

Pharmaceuticals are an important group of emerging pollutants with increasing interest due to their rising consumption and the evidence for ecotoxicological effects associated to trace amounts in aquatic environments. In this paper, we assessed the potential degradation of a series of pharmaceuticals: antibiotics (sulfamethoxazole), antidepressives (citalopram hydrobromide and fluoxetine hydrochloride), antiepileptics (carbamazepine), anti-inflammatory drugs (diclofenac and naproxen) and estrogen hormones (estrone, 17β-estradiol, 17α-ethinylestradiol) by means of a versatile peroxidase (VP) from the ligninolytic fungus Bjerkandera adusta. The effects of the reaction conditions: VP activity, organic acid concentration and H(2)O(2) addition rate, on the kinetics of the VP based oxidation system were evaluated. Diclofenac and estrogens were completely degraded after only 5-25 min even with a very low VP activity (10 U l(-1)). High degradation percentages (80%) were achieved for sulfamethoxazole and naproxen. Low or undetectable removal yields were observed for citalopram (up to 18%), fluoxetine (lower than 10%) and carbamazepine (not degraded).

Mutual influence of soil basidiomycetes and white mustard plants on their enzymatic and catabolic activities

Liquid and volatile emanations in interactions of soil basidiomycetes with herbs affect fungal oxidoreductases and stress-related plant peroxidases (PO). In this study, gnotobiotic co-cultures between 6 non-pathogenic saprobes and 2 ectomycorrhizal basidiomycetes with the non-host plant white mustard were established on glucose-salt medium with the respective controls. Determined were oxidoreductase activities for culture fluids and plant tissues at initial fungal idiophase and degradation rates of Remazol-BBR and 5 PAHs. In culture fluids of Agaricus arvensis, A. porphyrizon, Lepista nebularis, Stropharia rugoso-annulata, and Hypholoma fasciculare (group-5), the laccase-deficient plant enabled activity increases in fungal laccase (by 2300-fold), in extracellular (fungal and?) plant-derived peroxidases (by 21-fold), and in the dissipation of phenanthrene and anthracene. Oxidative activities in roots rose by 46000-fold during adsorption of fungal laccases. Increases in the stress-related shoot-PO (by 4.1-fold) were exclusively elicited by group-5 saprobes and correlated with plant-phenolic-mediated formations of Mn(III) and increases in Remazol BBR bleaching. Agaricus bisporus and the ectomycorrhizal Hebeloma crustuliniforme and Suillus granulatus did not respond to plant emanations with elevated laccase activities but solubilized apparently root-surface PO. They failed to elicit stress-related activity increases of PO in white mustard shoot and prevented Mn(III) formation in several tissues. It is concluded that white mustard emanations promoted the catabolic performance of the plant-stress eliciting group-5 saprobes but not of A. bisporus and the ectomycorrhizal fungi with their low stress-inducing potential. The nature of the plant-released stimuli and the classes of fungus-released stress agents discussed must be determined in further studies.

New and classic families of secreted fungal heme peroxidases

Heme-containing peroxidases secreted by fungi are a fascinating group of biocatalysts with various ecological and biotechnological implications. For example, they are involved in the biodegradation of lignocelluloses and lignins and participate in the bioconversion of other diverse recalcitrant compounds as well as in the natural turnover of humic substances and organohalogens. The current review focuses on the most recently discovered and novel types of heme-dependent peroxidases, aromatic peroxygenases (APOs), and dye-decolorizing peroxidases (DyPs), which catalyze remarkable reactions such as peroxide-driven oxygen transfer and cleavage of anthraquinone derivatives, respectively, and represent own separate peroxidase superfamilies. Furthermore, several aspects of the "classic" fungal heme-containing peroxidases, i.e., lignin, manganese, and versatile peroxidases (LiP, MnP, and VP), phenol-oxidizing peroxidases as well as chloroperoxidase (CPO), are discussed against the background of recent scientific developments.

Delignification of eucalypt kraft pulp with manganese-substituted polyoxometalate assisted by fungal versatile peroxidase

Oxidation of the manganese-substituted polyoxometalate [SiW11MnII(H2O)O39]6− (SiW11MnII) to [SiW11MnIII(H2O)O39]5− (SiW11MnIII), one of the most selective polyoxometalates for the kraft pulp delignification, by versatile peroxidase (VP) was studied. First, SiW11MnII was demonstrated to be quickly oxidized by VP at room temperature in the presence of H2O2 (Km=6.4±0.7mM and kcat=47±2s−1). Second, the filtrate from eucalypt pulp delignification containing reduced polyoxometalate was treated with VP/H2O2, and 95–100% reoxidation was attained. In this way, it was possible to reuse the liquor from a first SiW11MnIII stage for further delignification, in a sequence constituted by two polyoxometalate stages, and a short intermediate step consisting of the addition of VP/H2O2 to the filtrate for SiW11MnII reoxidation. When the first ClO2 stage of a conventional bleaching sequence was substituted by the two-stage delignification with polyoxometalate (assisted by VP) a 50% saving in ClO2 was obtained for similar mechanical strength of the final pulp.

Statistical analysis for enzymatic decolorization of acid orange 7 by Coprinus cinereus peroxidase

Enzymatic decolorization of the monoazo dye, acid orange 7 (AO7) by the fungal peroxidase from Coprinus cinereus NBRC 30628 is a complex system, which is greatly affected by temperature, pH, enzyme activity and the concentrations of H 2O 2 and dye concentration. The study of these factors and investigating the combined interactions between them by applying one-factor-at-a-time (OFAT) method and two other statistical methods including 2-factorial method and response surface methodology (RSM) were aimed in this work. Through OFAT analysis the optimized conditions were a temperature of 25 °C, pH 9.0 with H 2O 2 concentration of about 3.9 mM and AO7 concentration of 40 mg/l. A complete decolorization attained under such conditions in only 1 min. For statistical optimization, 2-factorial design method was applied first, and three factors of temperature, pH and initial dye concentration were recognized as significant factors. Then, response surface analysis was conducted through central composite design and a second-order polynomial model ( R 2 = 0.9718) was generated. ANOVA analysis of this model indicated that the temperature, pH and their interaction were the most significant in decolorization of AO7. The optimum condition through RSM was found to be the temperature of 25 °C, pH value of 9.0 and 50 mg/l of AO7 initial concentration, for the maximum decolorization of 100%.

Molecular characterization of lignin peroxidase from the white-rot basidiomycete Trametes cervina: a novel fungal peroxidase

The lignin peroxidase (LiP) from Trametes cervina was cloned, characterized, and identified as a novel fungal peroxidase. The sequence of T. cervina LiP encodes the essential amino acids for shaping the heme cavity and calcium-binding sites, which are conserved in plant and fungal peroxidases. However, a sequence homology analysis showed that T. cervina LiP has two unique features: it lacks the conserved tryptophan residue corresponding to the substrate-oxidation site (Trp171) of Phanerochaete chrysosporium LiP and it has a tyrosine residue (Tyr181) that has never been reported in other lignin peroxidases. A tertiary model of T. cervina LiP showed that Tyr181 sterically adjacent to the 6-propionate group of heme is surrounded by acidic amino acids and is exposed to the exterior. These attributes indicate that Tyr181 could be a T. cervina LiP substrate-oxidation site. A phylogenetic analysis showed that T. cervina LiP does not cluster with any other fungal peroxidases, suggesting that it is a unique molecule that is evolutionarily distant from other peroxidases. Thus, we concluded that T. cervina LiP could be a novel secreted peroxidase, among those produced by fungi, with a new oxidation mechanism probably involving Tyr181.

Enzyme activities and decolourization of single and mixed azo dyes by the white-rot fungus Phanerochaete chrysosporium

Decolourization of two azo dyes, namely Direct Red–80 (DR–80) and Mordant Blue–9 (MB–9), by Phanerochaete chrysosporium was investigated both individually and in mixtures in batch shake flasks. Mixture experiments containing these dyes were performed at five different initial concentrations ranging from 10 to 200 mg l −1 each. While the results of dye decolourization in mixture indicated decolourization efficiencies of more than 92%, the efficiencies were more than 94% in the single dye system. During the experiments, enzyme activities of lignin peroxidase (LiP) and manganese peroxidase (MnP) were also analyzed. Maximum LiP and MnP activities of 136 U l −1 and 16 U l −1, respectively, were achieved after 24 h of the fungal culture for respective initial DR–80 concentrations of 10 mg l −1 and 150 mg l −1 in the single dye system. In case of MB–9, maximum enzyme activities were 118 U l −1 (LiP) and 34 U l −1 (MnP) at initial concentrations of 50 mg l −1 and 100 mg l −1, respectively. When the two dyes were added together to the culture, maximum LiP and MnP activities were found to be 139 U l −1 and 132 U l −1 respectively for an initial concentration combination of 50 mg l −1 DR–80 and 10 mg l −1 MB–9. These profiles of enzyme activities and dye decolourization by the fungus, in both single and mixed dye systems, suggest the fungal peroxidase enzymes play a strong role in dye decolourization.

Generation of Norisoprenoid Flavors from Carotenoids by Fungal Peroxidases

To biotechnologically produce norisoprenoid flavor compounds, two extracellular peroxidases (MsP1 and MsP2) capable of degrading carotenoids were isolated from the culture supernatants of the basidiomycete Marasmius scorodonius (garlic mushroom). The encoding genes were cloned from genomic DNA and cDNA libraries, and databank homology searches identified MsP1 and MsP2 as members of the so-called "DyP-type" peroxidase family. Wild type enzymes and recombinant peroxidases expressed in Escherichia coli were employed for the release of norisoprenoids from various terpenoid precursor molecules. Carotenes, xanthophylls, and apocarotenals were subjected to the enzymatic degradation. Released volatile products were characterized by GC-FID and GC-MS, whereas nonvolatile breakdown products were analyzed by means of HPLC-DAD and HPLC-MS. C13 norisoprenoids together with C10 products proved to be the main volatile degradation products in each case.

Oxidizability of unsaturated fatty acids and of a non-phenolic lignin structure in the manganese peroxidase-dependent lipid peroxidation system

Unsaturated fatty acids have been proposed to mediate the oxidation of recalcitrant, non-phenolic lignin structures by fungal manganese peroxidases (MnP), but their precise role remains unknown. We investigated the oxidizability of three fatty acids with varying degrees of polyunsaturation (linoleic, linolenic, and arachidonic acids) by measuring conjugated dienes formation when lipid peroxidation was initiated either by MnP in the presence of Mn(II) or by chelated Mn(III). An inverse relationship between the degree of fatty acid unsaturation and the rate of peroxidation was found in both cases, but we also noted some differences between the two types of reaction. With MnP/Mn(II), the reaction developed slowly and resulted in sustained lipid peroxidation as determined by the formation of late-stage fatty acid degradation products. By contrast, the reaction with chelated Mn(III) was very rapid and did not result in the formation of these late-stage products, which suggests that this system failed to propagate the sustained radical chain reaction that is characteristic of complete lipid peroxidation. All three polyunsaturated fatty acids supported the co-oxidation of a non-phenolic lignin model compound by MnP, again showing an inverse relationship between the degree of unsaturation and reactivity, but chelated Mn(III) by itself supported only very low levels of fatty acid-mediated lignin model oxidation. These parallels in fatty acid reactivity are consistent with a reaction scheme in which Mn(III) acts as the proximal oxidant that initiates lipid peroxidation by MnP, thus generating fatty acid-derived radicals which in turn oxidize lignin structures. However, the results also suggest that the initial peroxyl radicals formed may not be the ligninolytic oxidants in this system. Instead, other radical oxidants produced during late-stage reactions of lipid peroxidation may be required.

ClassII peroxidase-encoding genes are present in a phylogenetically wide range of ectomycorrhizal fungi

Fungal peroxidases (ClassII) have a key role in degrading recalcitrant polyphenolic compounds in boreal forest wood, litter and humus. To date, their occurrence and activity have mainly been studied in a small number of white-rot wood decomposers. However, peroxidase activity is commonly measured in boreal forest humus and mineral soils, in which ectomycorrhizal fungi predominate. Here, we used degenerate PCR primers to investigate whether peroxidase-encoding genes are present in the genomes of a wide phylogenetic range of ectomycorrhizal taxa. Cloning and sequencing of PCR products showed that ectomycorrhizal fungi from several different genera possess peroxidase genes. The new sequences represent four major homobasidiomycete lineages, but the majority is derived from Cortinarius, Russula and Lactarius. These genera are ecologically important, but consist mainly of non-culturable species from which little ecophysiological information is available. The amplified sequences contain conserved active sites, both for folding and substrate oxidation. In some Cortinarius spp., there is evidence for gene duplications during the evolution of the genus. ClassII peroxidases seem to be an ancient and a common feature of most homobasidiomycetes, including ectomycorrhizal fungi. Production of extracellular peroxidases may provide ectomycorrhizal fungi with access to nitrogen sequestered in complex polyphenolic sources.

Curing behavior of polycardanol by MEKP and cobalt naphthenate using differential scanning calorimetry

In this study, polycardanol, which was synthesized by enzymatic oxidative polymerization of thermally treated cashew nut shell liquid (CNSL) using fungal peroxidase, was partially or fully cured using methyl ethyl ketone peroxide (MEKP) as initiator and cobalt naphthenate (Co-Naph) as accelerator. The curing behavior of polycardanol was extensively investigated in terms of curing temperature, curing time, concentration of initiator and accelerator, and the monomer-to-polymer conversion of polycardanol by means of differential scanning calorimetry (DSC). The curing behavior significantly depends on the thermal condition given and it was monitored with the change of the exotherms as a function of temperature. The optimal conditions for fully curing polycardanol are 1 wt% MEKP, 0.2 wt% Co-Naph, curing time 120 min, and curing temperature 200 °C. This study suggests that a polycardanol with high monomer-to-polymer conversion would be useful for processing a polycardanol matrix composite under the optimal conditions of curing.

Escherichia coli expression and in vitro activation of a unique ligninolytic peroxidase that has a catalytic tyrosine residue

Heterologous expression of Trametes cervina lignin peroxidase (LiP), the only basidiomycete peroxidase that has a catalytic tyrosine, was investigated. The mature LiP cDNA was cloned into the pET vector and used to transform Escherichia coli. Recombinant LiP protein accumulated in inclusion bodies as an inactive form. Refolding conditions for its in vitro activation—including incorporation of heme and structural Ca 2+ ions, and formation of disulfide bridges—were optimized taking as a starting point those reported for other plant and fungal peroxidases . The absorption spectrum of the refolded enzyme was identical to that of wild LiP from T. cervina suggesting that it was properly folded. The enzyme was able to oxidize 1,4-dimethoxybenzene and ferrocytochrome c confirming its high redox potential and ability to oxidize large substrates. However, during oxidation of veratryl alcohol (VA), the physiological LiP substrate, an unexpected initial lag period was observed. Possible modification of the enzyme was investigated by incubating it with H 2O 2 and VA (for 30 min before dialysis). The pretreated enzyme showed normal kinetics traces for VA oxidation, without the initial lag previously observed. Steady-state kinetics of the pretreated LiP were almost the same as the recombinant enzyme before the pretreatment. Moreover, the catalytic constant ( k cat) for VA oxidation was comparable to that of wild LiP from T. cervina, although the Michaelis–Menten constant ( K m) was 8-fold higher. The present heterologous expression system provides a valuable tool to investigate structure–function relationships, and autocatalytic activation of the unique T. cervina LiP.

Molecular characterization of aromatic peroxygenase from Agrocybe aegerita

Recently, a novel group of fungal peroxidases, known as the aromatic peroxygenases (APO), has been discovered. Members of these extracellular biocatalysts produced by agaric basidiomycetes such as Agrocybe aegerita or Coprinellus radians catalyze reactions--for example, the peroxygenation of naphthalene, toluene, dibenzothiophene, or pyridine--which are actually attributed to cytochrome P450 monooxygenases. Here, for the first time, genetic information is presented on this new group of peroxide-consuming enzymes. The gene of A. aegerita peroxygenase (apo1) was identified on the level of messenger RNA and genomic DNA. The gene sequence was affirmed by peptide sequences obtained through an Edman degradation and de novo peptide sequencing of the purified enzyme. Quantitative real-time reverse transcriptase polymerase chain reaction demonstrated that the course of enzyme activity correlated well with that of mRNA signals for apo1 in A. aegerita. The full-length sequences of A. aegerita peroxygenase as well as a partial sequence of C. radians peroxygenase confirmed the enzymes' affiliation to the heme-thiolate proteins. The sequences revealed no homology to classic peroxidases, cytochrome P450 enzymes, and only little homology (<30%) to fungal chloroperoxidase produced by the ascomycete Caldariomyces fumago (and this only in the N-terminal part of the protein comprising the heme-binding region and part of the distal heme pocket). This fact reinforces the novelty of APO proteins. On the other hand, homology retrievals in genetic databases resulted in the identification of various APO homologous genes and transcripts, particularly among the agaric fungi, indicating APO's widespread occurrence in the fungal kingdom.

Glucose exerts a negative effect over a peroxidase from Trichosporon asahii, with carotenoid cleaving activity

Tobacco aroma compounds were generated via lutein cleavage by the combined action of a yeast and a bacterium identified as Trichosporon asahii and Paenibacillus amylolyticus, respectively. In this study, an inverse relationship between glucose concentration and the generation of three compounds, present in the tobacco aroma profile, was observed in mixed cultures. In order to identify the organism sensitive to the sugar effect, both were grown separately. The presence of glucose suppressed beta-ionone production by T. asahii grown with lutein. However, the biotransformation of the ionone into its reduced derivatives (7,8-dihydro-beta-ionone and 7,8-dihydro-beta-ionol) by P. amylolyticus was not affected by the sugar. This pointed to the cleavage of lutein, a step within the process necessary for the synthesis of beta-ionone, as the target of the glucose effect. In vitro studies with crude extracts and concentrated cell-free medium derived from T. asahii cultures showed that the carotenoid breakdown activity was located extracellularly and only detected in supernatants from yeast cells grown in the absence of the sugar. Rather than an inhibition or a mechanism affecting the enzyme secretion, the glucose effect on lutein degradation comprised another regulatory level. Further experiments showed that the enzyme responsible for lutein breakdown and susceptible to the sugar effect exhibited a high degree of identity to fungal peroxidases, studied as well, for their involvement in carotenoid cleavage.

Increased PCP removal by Amylomyces rouxii transformants with heterologous Phanerochaete chrysosporium peroxidases supplementing their natural degradative pathway

Fungal peroxidases and phenoloxidases are widely used in aromatic toxic compounds degradation. Peroxidases, such as lignin peroxidase and manganese peroxidase, as well as laccases are mainly produced by basidiomycetes and to a lower extent by other fungi, such as ascomycetes. Peroxidase-encoding genes have been described and homologous expression has been achieved in basidiomycetes. Heterologous expression has also been achieved in some non-producing peroxidase ascomycetes, like Penicillium and Aspergillus. In this work, heterologous expression of peroxidase-encoding genes, lignin peroxidase, and manganese peroxidase was achieved in a zygomycete producing only phenoloxidases (Amylomyces rouxii), aimed at coupling two different pathways used in nature for PCP removal in only one microbial strain. The ability of PCP removal was assayed with one of the obtained transformants, resulting in increased activity with respect to the ability of the parental strain cultured free of the inducer tyrosine (95% and 45%, respectively, of the initial PCP (12.5 mg L(-1)) in 120 h, or 100% and 49%, respectively, of the initial PCP after 144 h of liquid culture).

Protein Radicals in Fungal Versatile Peroxidase

Lignin-degrading peroxidases, a group of biotechnologically interesting enzymes, oxidize high redox potential aromatics via an exposed protein radical. Low temperature EPR of Pleurotus eryngii versatile peroxidase (VP) revealed, for the first time in a fungal peroxidase, the presence of a tryptophanyl radical in both the two-electron (VPI) and the one-electron (VPII) activated forms of the enzyme. Site-directed mutagenesis was used to substitute this tryptophan (Trp-164) by tyrosine and histidine residues. No changes in the crystal structure were observed, indicating that the modified behavior was due exclusively to the mutations introduced. EPR revealed the formation of tyrosyl radicals in both VPI and VPII of the W164Y variant. However, no protein radical was detected in the W164H variant, whose VPI spectrum indicated a porphyrin radical identical to that of the inactive W164S variant. Stopped-flow spectrophotometry showed that the W164Y mutation reduced 10-fold the apparent second-order rate constant for VPI reduction (k(2app)) by veratryl alcohol (VA), when compared with over 50-fold reduction in W164S, revealing some catalytic activity of the tyrosine radical. Its first-order rate constant (k(2)) was more affected than the dissociation constant (K(D)(2)). Moreover, VPII reduction by VA was impaired by the above mutations, revealing that the Trp-164 radical was involved in catalysis by both VPI and VPII. The low first-order rate constant (k(3)) values were similar for the W164Y, W164H, and W164S variants, indicating that the tyrosyl radical in VPII was not able to oxidize VA (in contrast with that observed for VPI). VPII self-reduction was also suppressed, revealing that Trp-164 is involved in this autocatalytic process.

Transgenic plants and associated bacteria for phytoremediation of chlorinated compounds

Phytoremediation is the use of plants for the treatment of environmental pollution, including chlorinated organics. although conceptually very attractive, removal and biodegradation of chlorinated pollutants by plants is a rather slow and inefficient process resulting in incomplete treatment and potential release of toxic metabolites into the environment. In order to overcome inherent limitations of plant metabolic capabilities, plants have been genetically modified, following a strategy similar to the development of transgenic crops: genes from bacteria, fungi, and mammals involved in the metabolism of organic contaminants, such as cytochrome p-450 and glutathione substrate catabolic genes, natural or engineered, for the simultaneous remediation of a range of pollutants, such as usually found in contaminated sites, e.g., chlorinated solvent, metals, and nitroaromatics. In addition, biodegradation of many xenobiotics are catalyzed by similar, broad-substrate enzymes, such as cytochrome P-450 monoxygenases, glutathione S-transferases, and fungal peroxidases, that can potentially be used for the treatment of multiple pollutants. Moreover, the introduction of multiple transgenes involved in different phases of the metabolism of xenobiotics in plants, i.e., uptake by roots and the different phases of the green liver model, would allow enhancing both the removal and metabolism of several toxic compounds and could therefore help overcome a major limitation inherent to phytoremediation, i.e., the threat that accumulated toxic compounds would volatilize or otherwise contaminate the food chain. An important barrier to the application of transgenic plants for bioremediation in the field is associated with the true or perceived risk of horizontal gene transfer to related wild or cultivated plants. Therefore, it is likely that the next generation of transgenic plants will involve systems preventing such a transfer, for instance by the introduction of transgenes into chloroplastic DNA or the use of conditional lethality genes (Davison, 2005). Since bacteria naturally exchange plasmids via conjugation, endophytes that gain genes involved in pollutant degradation might not be considered ‘genetically modified’ and may be subject to fewer restrictions in usage.